Materials and methods for protein production

ABSTRACT

This document relates to materials and methods for the production of protein. In one aspect, this document provides a nucleic acid construct including a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element includes a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to SEQ ID NO: 28.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/835,338, filed on Apr. 17, 2019, which is incorporated byreference herein in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing filename: 38767_0193001_SEQ.txt,date recorded, Apr. 17, 2020, file size 53 kilobytes.

TECHNICAL FIELD

This disclosure generally relates to DNA constructs and methods of usingsuch DNA constructs to genetically engineer cells, such as yeast cellsor methylotrophic yeast cells.

BACKGROUND

Recombinant expression of products is a common method to produce saidproducts. In some cases, proteins can be produced by recombinantproduction. Constructs that can be used to efficiently express one ormore products (e.g., proteins) in a cell, such as a yeast cell or amethylotrophic yeast cell, are provided herein.

SUMMARY

This document is based, at least in part, on the identification of pointmutations in the AOX1 promoter that can confer increased expression oflinked coding sequences. The mutated AOX1 promoters described herein canbe used for efficient expression of operably linked coding sequences inPichia , for example.

In one aspect, provided herein is a nucleic acid construct comprising afirst alcohol oxidase promoter element, wherein the first alcoholoxidase promoter element includes a mutation at one or more nucleotidepositions corresponding to any of nucleotide positions 668-734 relativeto SEQ ID NO: 28.

Implementations can have one or more of the following features. Thefirst alcohol oxidase promoter element can include a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions673-729 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include a mutation at one or more nucleotide positionscorresponding to any of nucleotide positions 678-724 relative to SEQ IDNO: 28. The first alcohol oxidase promoter element can include amutation at one or more nucleotide positions corresponding to any ofnucleotide positions 683-719 relative to SEQ ID NO: 28. The firstalcohol oxidase promoter element can include a mutation at one or morenucleotide positions corresponding to any of nucleotide positions688-714 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include two or more mutations at nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO: 28. The first alcohol oxidase promoter element can include three ormore mutations at nucleotide positions corresponding to any ofnucleotide positions 668-734 relative to SEQ ID NO: 28. The firstalcohol oxidase promoter element can include four or more mutations atnucleotide positions corresponding to any of nucleotide positions668-734 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include five or more mutations at nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO: 28.

In another aspect, provided herein is a nucleic acid constructcomprising a first alcohol oxidase promoter element, wherein the firstalcohol oxidase promoter element can include one or more mutations at anucleotide position selected from the group consisting of nucleotidepositions corresponding to T146, C154, T303, T426, A433, A435, T530,C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, andT862 relative to SEQ ID NO: 28.

Implementations can include one or more of the following features. Thefirst alcohol oxidase promoter element can include two or more mutationsat nucleotide positions selected from the group consisting of nucleotidepositions corresponding to T146, C154, T303, T426, A433, A435, T530,C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, andT862 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include three or more mutations at nucleotide positionsselected from the group consisting of nucleotide positions correspondingto T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688,A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ IDNO: 28. The first alcohol oxidase promoter element can include four ormore mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to T146, C154, T303,T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712,T714, A790, A841, and T862 relative to SEQ ID NO: 28. The first alcoholoxidase promoter element can include five or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to T146, C154, T303, T426, A433, A435, T530,C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, andT862 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include one or more mutations at a nucleotide positionselected from the group consisting of nucleotide positions correspondingto T688, A696, T702, A712, and T714 relative to SEQ ID NO: 28. The firstalcohol oxidase promoter element can include two or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to T688, A696, T702, A712, and T714 relative toSEQ ID NO: 28. The first alcohol oxidase promoter element can includethree or more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to T688, A696, T702,A712, and T714 relative to SEQ ID NO: 28. The first alcohol oxidasepromoter element can include four or more mutations at nucleotidepositions selected from the group consisting of nucleotide positionscorresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO:28. The first alcohol oxidase promoter element can include mutations atnucleotide positions corresponding to T688, A696, T702, A712, and T714relative to SEQ ID NO: 28.

In another aspect, provided herein is a nucleic acid constructcomprising a first alcohol oxidase promoter element, wherein the firstalcohol oxidase promoter element can include one or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C,A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative toSEQ ID NO: 28.

Implementations can include one or more of the following features. Thefirst alcohol oxidase promoter element can include two or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T,

T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T,and T862A relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include three or more mutations selected from the groupconsisting of mutations corresponding to T146C, C154T, T303C, T426A,A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G,A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28. Thefirst alcohol oxidase promoter element can include four or moremutations selected from the group consisting of mutations correspondingto T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C,T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862Arelative to SEQ ID NO: 28. The first alcohol oxidase promoter elementcan include five or more mutations selected from the group consisting ofmutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G,T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G,A790G, A841T, and T862A relative to SEQ ID NO: 28. The first alcoholoxidase promoter element can include one or more mutations selected fromthe group consisting of T688C, A696T, T702C, A712G, and T714G relativeto SEQ ID NO: 28. The first alcohol oxidase promoter element can includetwo or more mutations selected from the group consisting of T688C,A696T, T702C, A712G, and T714G relative to SEQ ID NO: 28. The firstalcohol oxidase promoter element can include three or more mutationsselected from the group consisting of T688C, A696T, T702C, A712G, andT714G relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include four or more mutations selected from the groupconsisting of T688C, A696T, T702C, A712G, and T714G relative to SEQ IDNO: 28. The first alcohol oxidase promoter element can include themutations T688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO:28.

Implementations of any of the nucleic acid constructs described hereincan have one or more of the following features. The first alcoholoxidase promoter element can be an alcohol oxidase 1 promoter element.The first alcohol oxidase promoter element can have at least 90%sequence identity to SEQ ID NO: 28. The first alcohol oxidase promoterelement can have at least 95% sequence identity to SEQ ID NO: 28. Thenucleic acid construct can further include a nucleotide sequenceencoding a first protein, wherein the nucleotide sequence encoding thefirst protein is operably linked to the first alcohol oxidase promoterelement. The first protein can be exogenous to a methylotrophic yeastcell. The first protein can be heterologous to a methylotrophic yeastcell. The first protein can be selected from the group consisting of anantibody or fragment thereof, an enzyme, a regulatory protein, a peptidehormone, a blood clotting protein, a cytokine, a cytokine inhibitor, anda heme-binding protein. The first protein can be a heme-binding protein.The heme-binding protein can be selected from the group consisting of aglobin, a cytochrome, a cytochrome c oxidase, a ligninase, a catalase,and a peroxidase. The heme-binding protein can be selected from thegroup consisting of an androglobin, a chlorocruorin, a cytoglobin, anerythrocruorin, a flavohemoglobin, a globin E, a globin X, a globin Y, ahemoglobin, a histoglobin, a leghemoglobin, a myoglobin, a neuroglobin,a non-symbiotic hemoglobin, a protoglobin, and a truncated hemoglobin.The heme-binding protein can be a non-symbiotic hemoglobin. Theheme-binding protein can be a leghemoglobin. The heme-binding proteincan include an amino acid sequence having at least 90% sequence identityto the amino acid sequence of any of SEQ ID NOs: 1-27. The first alcoholoxidase promoter element can include a recognition sequence for atranscription factor.

In another aspect, also provided herein is a methylotrophic yeast cellcomprising a first nucleic acid construct, wherein the first nucleicacid construct is any nucleic acid construct described herein.

Implementations can have one or more of the following features. Themethylotrophic yeast cell can be a Pichia cell, a Candida cell, aHansenula cell, or a Torulopsis cell. The methylotrophic yeast cell canbe a Pichia methanolica cell, a Pichia pastoris cell, a Candida boidiniicell, or a Hansenula polymorpha cell. The methylotrophic yeast cell canbe a Pichia pastoris cell. The methylotrophic yeast cell can furtherinclude a second nucleic acid construct including a nucleotide sequenceencoding a second protein, wherein the nucleotide sequence encoding thesecond protein is operably linked to the first alcohol oxidase promoterelement or to a second promoter element. The nucleotide sequenceencoding the second protein can be operably linked to a second promoterelement that has the same sequence as the first alcohol oxidase promoterelement. The second protein can be a transcription factor. Thenucleotide sequence encoding the second protein can be operably linkedto a second promoter element that can include a recognition sequence forthe transcription factor. The first alcohol oxidase promoter element caninclude a recognition sequence for the transcription factor. The secondprotein can be a protein involved in heme biosynthesis. The proteininvolved in heme biosynthesis can be selected from the group consistingof aminolevulinic acid synthase (ALAS), δ-aminolevulinic aciddehydratase (ALAD), porphogilinogen deaminase (PBGD), uroporphyrinogenIII synthase (UPG3 S), uroporphyrinogen III decarboxylase (UPG3D),coprotoporphyrinogen oxidase (COPROX), protoporphyrinogen IX oxidase(PROTOX), and ferrochelatase (FC).

In another aspect, provided herein is method of producing a protein in amethylotrophic yeast cell including expressing a nucleic acid constructincluding a nucleotide sequence encoding a first protein operably linkedto a first alcohol oxidase promoter element, wherein the first alcoholoxidase promoter element includes a mutation at one or more nucleotidepositions corresponding to any of nucleotide positions 668-734 relativeto SEQ ID NO: 28.

Implementations can include one or more of the following features. Thefirst alcohol oxidase promoter element can include a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions673-729 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include a mutation at one or more nucleotide positionscorresponding to any of nucleotide positions 678-724 relative to SEQ IDNO: 28. The first alcohol oxidase promoter element can include amutation at one or more nucleotide positions corresponding to any ofnucleotide positions 683-719 relative to SEQ ID NO: 28. The firstalcohol oxidase promoter element can include a mutation at one or morenucleotide positions corresponding to any of nucleotide positions688-714 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include two or more mutations at nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO: 28. The first alcohol oxidase promoter element can include three ormore mutations at nucleotide positions corresponding to any ofnucleotide positions 668-734 relative to SEQ ID NO: 28. The firstalcohol oxidase promoter element can include four or more mutations atnucleotide positions corresponding to any of nucleotide positions668-734 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include five or more mutations at nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO: 28.

In another aspect, also provided herein is a method of producing aprotein in a methylotrophic yeast cell including expressing a nucleicacid construct including a nucleotide sequence encoding a first proteinoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element includes one or more mutations ata nucleotide position selected from the group consisting of nucleotidepositions corresponding to T146, C154, T303, T426, A433, A435, T530,C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, andT862 relative to SEQ ID NO: 28.

Implementations can include one or more of the following features. Thefirst alcohol oxidase promoter element can include two or more mutationsat nucleotide positions selected from the group consisting of nucleotidepositions corresponding to T146, C154, T303, T426, A433, A435, T530,C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, andT862 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include three or more mutations at nucleotide positionsselected from the group consisting of nucleotide positions correspondingto T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688,A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ IDNO: 28. The first alcohol oxidase promoter element can include four ormore mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to T146, C154, T303,T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712,T714, A790, A841, and T862 relative to SEQ ID NO: 28. The first alcoholoxidase promoter element can include five or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to T146, C154, T303, T426, A433, A435, T530,C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, andT862 relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include one or more mutations at a nucleotide positionselected from the group consisting of nucleotide positions correspondingto of T688, A696, T702, A712, and T714 relative to SEQ ID NO: 28. Thefirst alcohol oxidase promoter element can include two or more mutationsat nucleotide positions selected from the group consisting of nucleotidepositions corresponding to of T688, A696, T702, A712, and T714 relativeto SEQ ID NO: 28. The first alcohol oxidase promoter element can includethree or more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to of T688, A696, T702,A712, and T714 relative to SEQ ID NO: 28. The first alcohol oxidasepromoter element can include four or more mutations at nucleotidepositions selected from the group consisting of nucleotide positionscorresponding to of T688, A696, T702, A712, and T714 as compared to SEQID NO: 28. The first alcohol oxidase promoter element can includemutations at nucleotide positions corresponding to T688, A696, T702,A712, and T714 as compared to SEQ ID NO: 28.

In another aspect, provided herein is a method of producing a protein ina methylotrophic yeast cell including expressing a nucleic acidconstruct including a nucleotide sequence encoding a first proteinoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element includes one or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C,A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative toSEQ ID NO: 28.

Implementations can include one or more of the following features. Thefirst alcohol oxidase promoter element can include two or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C,A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative toSEQ ID NO: 28. The first alcohol oxidase promoter element can includethree or more mutations selected from the group consisting of mutationscorresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T,T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T,and T862A relative to SEQ ID NO: 28. The first alcohol oxidase promoterelement can include four or more mutations selected from the groupconsisting of mutations corresponding to T146C, C154T, T303C, T426A,A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G,A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28. Thefirst alcohol oxidase promoter element can include five or moremutations selected from the group consisting of mutations correspondingto T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C,T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862Arelative to SEQ ID NO: 28. The first alcohol oxidase promoter elementcan include two or more mutations selected from the group consisting ofT688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO: 28. Thefirst alcohol oxidase promoter element can include three or moremutations selected from the group consisting of T688C, A696T, T702C,A712G, and T714G relative to SEQ ID NO: 28. The first alcohol oxidasepromoter element can include four or more mutations selected from thegroup consisting of T688C, A696T, T702C, A712G, and T714G relative toSEQ ID NO: 28. The first alcohol oxidase promoter element can includethe mutations T688C, A696T, T702C, A712G, and T714G relative to SEQ IDNO: 28.

Implementations of any of the methods described herein can have one ormore of the following features. The first alcohol oxidase promoterelement can be an alcohol oxidase 1 promoter element. The first alcoholoxidase promoter element can have at least 90% sequence identity to SEQID NO: 28. The first alcohol oxidase promoter element can have at least95% sequence identity to SEQ ID NO: 28. The first protein can beexogenous to the methylotrophic yeast cell. The first protein can beheterologous to the methylotrophic yeast cell. The first protein can beselected from the group consisting of an antibody or fragment thereof,an enzyme, a regulatory protein, a peptide hormone, a blood clottingprotein, a cytokine, and a heme-binding protein. The first protein canbe a heme-binding protein. The heme-binding protein can be selected fromthe group consisting of a globin, a cytochrome, a cytochrome c oxidase,a ligninase, a catalase, and a peroxidase. The heme-binding protein anbe selected from the group consisting of an androglobin, achlorocruorin, a cytoglobin, an erythrocruorin, a flavohemoglobin, aglobin E, a globin X, a globin Y, a hemoglobin, a histoglobin, aleghemoglobin, a myoglobin, a neuroglobin, a non-symbiotic hemoglobin, aprotoglobin, and a truncated hemoglobin. The heme-binding protein can bea non-symbiotic hemoglobin. The heme-binding protein can be aleghemoglobin. The heme-binding protein can include an amino acidsequence having at least 90% sequence identity to an amino acid sequencein any one of SEQ ID NOs: 1-27. The first alcohol oxidase promoterelement can contain one or more recognition sequences for atranscription factor. The method can further include expressing a secondnucleic acid construct including a nucleotide sequence encoding a secondprotein, wherein the nucleotide sequence encoding the second protein isoperably linked to the first alcohol oxidase promoter element or to asecond promoter element. The nucleotide sequence encoding the secondprotein can be operably linked to a second promoter element that has thesame sequence as the first alcohol oxidase promoter element. The secondprotein can be a transcription factor. The nucleotide sequence encodingthe second protein can be operably linked to a second promoter elementthat can include a recognition sequence for the transcription factor.The first alcohol oxidase promoter element can include a recognitionsequence for the transcription factor. The second protein can be aprotein involved in heme biosynthesis. The protein involved in hemebiosynthesis can be selected from the group consisting of ALAS, ALAD,PBGD, UPG3S, UPG3D, COPROX, PROTOX, and FC. The method can be carriedout in the absence of added methanol.

In another aspect, provided herein is a Pichia pastoris cell including anucleic acid construct comprising a nucleotide sequence encoding a firstalcohol oxidase promoter element, wherein the first alcohol oxidasepromoter element includes one or more mutations selected from the groupconsisting of mutations corresponding to T146C, C154T, T303C, T426A,A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G,A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28. In someembodiments, the one or more mutations can be selected from the groupconsisting of mutations corresponding to T688C, A696T, T702C, A712G, andT714G relative to SEQ ID NO: 28.

In another aspect, also provided herein is a method of producingleghemoglobin, the method including expressing a nucleic acid constructincluding a nucleotide sequence encoding leghemoglobin operably linkedto a first alcohol oxidase promoter element, wherein the first alcoholoxidase promoter element includes one or more mutations selected fromthe group consisting of mutations corresponding to T146C, C154T, T303C,T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C,A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28.In some embodiments, the method can be carried out in the absence ofadded methanol. In some embodiments, the one or more mutations can beselected from the group consisting of mutations corresponding to T688C,A696T, T702C, A712G, and T714G relative to SEQ ID NO: 28.

In another aspect, provided herein is a Pichia pastoris cell including afirst nucleic acid construct including a nucleotide sequence with atleast 90% sequence identity to SEQ ID NO: 28, wherein the first nucleicacid construct includes one or more mutations selected from the groupconsisting of mutations corresponding to T146C, C154T, T303C, T426A,A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G,A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28. In someembodiments, the one or more mutations can be selected from the groupconsisting of mutations corresponding to T688C, A696T, T702C, A712G, andT714G relative to SEQ ID NO: 28.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the sequences of exemplary heme-binding proteins (SEQ IDNOs: 1-27).

FIG. 2 provides the sequences of pAOX1 wild-type and mutant sequences(SEQ ID NOs: 28-29).

FIG. 3 is an image showing growth of pMx0414 transformants on YPDmedium.

FIG. 4 is a graph plotting the relative expression of GFP in strainsMxY0270 and MxY0279 under different growth conditions.

FIG. 5 is a comparison of portions of the sequence of MxG0038 andMxG0220.

FIG. 6 is a graph plotting the relative expression of GFP in strainsMxY0964, MxY965, and MxY1039.

FIG. 7 provides the sequences of SEQ ID NOs: 30-37.

DETAILED DESCRIPTION

This document is related to materials and methods for proteinproduction. For example, in one aspect, this document is related tomaterials and method for the production of products (e.g., proteins(e.g., plant proteins)) in cells (e.g., yeast (e.g., methylotrophicyeast) using an engineered promoter.

Methylotrophic yeast, such as Pichia pastoris, are commonly used toproduce recombinant products (e.g., proteins). Pichia strains aretypically able to grow on methanol as the sole carbon source. It will beunderstood that Pichia pastoris has been reclassified as Komagataellaspecies, such as Komagataella phaffii, Komagataella pastoris, orKomagataella pseudopastoris, though the term ‘Pichia pastoris’ is stillin use and may refer to any appropriate Komagataella species. Commonly,laboratory strains of P. pastoris are Komagataella phaffii.

Methanol utilization can be initiated by the conversion of methanol toformaldehyde by the action of alcohol oxidase. P. pastoris contains twogenes for alcohol oxidases, AOX1 and AOX2. Strains with reduced alcoholoxidase activity (“methanol utilization slow” or MutS strains) canusually produce more of a recombinant product (e.g., protein) expressedfrom the AOX1 promoter than strains that do not have reduced alcoholoxidase activity. The Pichia pastoris promoter for the alcohol oxidase 1(AOX1) gene, referred to as pAOX1, can be used for production ofheterologous products (e.g., proteins (e.g., proteins of industrialrelevance)). Expression from this promoter can be induced in thepresence of methanol, a flammable and toxic compound. In someembodiments, the materials and methods described herein can allow forexpression of recombinant products (e.g., proteins) at high level fromthis promoter, or a promoter element therefrom, in the absence ofmethanol. In some embodiments, the materials and methods describedherein can allow for expression of recombinant products (e.g., proteins)at high level from this promoter, or a promoter element therefrom, inthe absence of added methanol.

Expression from pAOX1 is typically absent or very poor in the presenceof non-inducing carbon sources, such as glucose or glycerol. Herein aredescribed mutations in pAOX1 that allow significant expression frompAOX1 in the absence of methanol. Herein are described mutations inpAOX1 that allow significant expression from pAOX1 in the absence ofadded methanol. A reference pAOX1 sequence is provided in SEQ ID NO: 28(FIG. 2). Exemplary mutations in pAOX1, as described herein, areprovided in SEQ ID NO: 29 (FIG. 2). These mutations can be presentindividually or in any combination. These mutations can also provide anadditional increase in expression from pAOX1 when methanol is present.

Thus, provided herein are nucleic acid constructs (sometimes also callednucleic acid molecules) that include a promoter element having asequence that includes one or more mutations as compared to a referencepromoter sequence. In some embodiments, a promoter element can be analcohol oxidase promoter element. In some embodiments, a promoterelement can have at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, or 99%) sequence identity to an alcohol oxidase promoterelement (e.g., SEQ ID NO: 28 or SEQ ID NO: 29). In some embodiments, apromoter element can have the sequence of SEQ ID NO: 29. In someembodiments, a single mutation can be present in a promoter element. Forexample, in some embodiments, a single mutation corresponding to amutation in one of nucleotide positions 668-734 (e.g., nucleotidepositions 673-729, nucleotide positions 678-724, nucleotide positions683-719, or nucleotide positions 688-714) relative to SEQ ID NO: 28 canbe present in a promoter element. For example, in some embodiments, asingle mutation corresponding to one of the following mutations relativeto SEQ ID NO: 28 can be present in a promoter element: T146C; C154T;T303C; T426A; A433T; A435G; T530A; C572T; T596C; T617C; T688C; A696T;T702C; A709G; A712G; T714G; A790G; A841T; or T862A. For example, in someembodiments, a single mutation corresponding to one of the followingmutations relative to SEQ ID NO: 28 can be present in a promoterelement: 146C; 154T; 303C; 426A; 433T; 435G; 530A; 572T; 596C; 617C;688C; 696T; 702C; 709G; 712G; 714G; 790G; 841T; or 862A, as long as theindicated nucleobase is not the same as the correspondingnaturally-occurring nucleobase. For example, in some embodiments, asingle mutation at a position corresponding to one of the followingpositions relative to SEQ ID NO: 28 can be present in a promoterelement: T146; C154; T303; T426; A433; A435; T530; C572; T596; T617;T688; A696; T702; A709; A712; T714; A790; A841; or T862. For example, insome embodiments, a single mutation at a position corresponding to oneof the following positions relative to SEQ ID NO: 28 can be present in apromoter element: 146; 154; 303; 426; 433; 435; 530; 572; 596; 617; 688;696; 702; 709; 712; 714; 790; 841; or 862. For example, in someembodiments, a single mutation corresponding to one of the followingmutations relative to SEQ ID NO: 28 can be present in a promoterelement: T688C; A696T; T702C; A712G; or T714G. For example, in someembodiments, a single mutation corresponding to one of the followingmutations relative to SEQ ID NO: 28 can be present in a promoterelement: 688C; 696T; 702C; 712G; or 714G, as long as the indicatednucleobase is not the same as the corresponding naturally-occurringnucleobase. For example, in some embodiments, a single mutation at aposition corresponding to one of the following positions relative to SEQID NO: 28 can be present in a promoter element: T688; A696; T702; A712;or T714. For example, in some embodiments, a single mutation at aposition corresponding to one of the following positions relative to SEQID NO: 28 can be present in a promoter element: 688; 696; 702; 712; or714.

Also provided herein are nucleic acid constructs that include a promoterelement having a sequence that includes multiple (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutationsas compared to a reference promoter sequence. For example, in someembodiments, at least 2 (e.g., at least 3, at least 4, at least 5, atleast 10, at least 15, 2 to 5, 2 to 10, 2 to 15, 2 to 20, 5 to 10, 5 to15, 5 to 20, 10 to 15, 10 to 20, or 15 to 20) mutations corresponding toa mutation in nucleotide positions 668-734 (e.g., nucleotide positions673-729, nucleotide positions 678-724, nucleotide positions 683-719, ornucleotide positions 688-714) relative to SEQ ID NO: 28 can be presentin a promoter element. For example, in some embodiments, at least 2(e.g., at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 12, at least 14, at least 16,at least 18, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,or 19) mutations corresponding to the following mutations relative toSEQ ID NO: 28 can be present in a promoter element: T146C; C154T; T303C;T426A; A433T; A435G; T530A; C572T; T596C; T617C; T688C; A696T; T702C;A709G; A712G; T714G; A790G; A841T; or T862A. For example, in someembodiments, at least 2 (e.g., at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 12,at least 14, at least 16, at least 18, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, or 19) mutations corresponding to thefollowing mutations relative to SEQ ID NO: 28 can be present in apromoter element: 146C; 154T; 303C; 426A; 433T; 435G; 530A; 572T; 596C;617C; 688C; 696T; 702C; 709G; 712G; 714G; 790G; 841T; or 862A, as longas the indicated nucleobase is not the same as the correspondingnaturally-occurring nucleobase. For example, in some embodiments, atleast 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, at least 10, at least 12, at least 14, atleast 16, at least 18, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, or 19) mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promoterelement: T146; C154; T303; T426; A433; A435; T530; C572; T596; T617;T688; A696; T702; A709; A712; T714; A790; A841; or T862. For example, insome embodiments, at least 2 (e.g., at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least12, at least 14, at least 16, at least 18, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations at positionscorresponding to the following positions relative to SEQ ID NO: 28 canbe present in a promoter element: 146; 154; 303; 426; 433; 435; 530;572; 596; 617; 688; 696; 702; 709; 712; 714; 790; 841; or 862. Forexample, in some embodiments, at least 2 (e.g., at least 3, at least 4,2, 3, 4, or 5) mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter element: T688C;A696T; T702C; A712G; or T714G. For example, in some embodiments, atleast 2 (e.g., at least 3, at least 4, 2, 3, 4, or 5) mutationscorresponding to the following mutations relative to SEQ ID NO: 28 canbe present in a promoter element: 688C; 696T; 702C; 712G; or 714G, aslong as the indicated nucleobase is not the same as the correspondingnaturally-occurring nucleobase. For example, in some embodiments, atleast 2 (e.g., at least 3, at least 4, 2, 3, 4, or 5) mutationscorresponding to one of the following positions relative to SEQ ID NO:28 can be present in a promoter element: T688; A696; T702; A712; orT714. For example, in some embodiments, at least 2 (e.g., at least 3, atleast 4, 2, 3, 4, or 5) mutations corresponding to one of the followingpositions relative to SEQ ID NO: 28 can be present in a promoterelement: 688; 696; 702; 712; or 714.

In some embodiments, a mutation in a nucleic acid can be an insertion, adeletion or a substitution. In some embodiments, a mutation in a nucleicacid can be a substitution (e.g., a guanosine to cytosine mutation). Insome embodiments, a mutation in a nucleic acid can be in a non-codingsequence. In some embodiments, a substitution in a coding sequence(e.g., encoding a protein) can be a silent mutation (e.g., the sameamino acid is encoded). In some embodiments, a substitution in a codingsequence can be a nonsynonymous mutation (e.g., a missense mutation or anonsense mutation). In some embodiments, a substitution in a codingsequence can be a missense mutation (e.g., a different amino acid isencoded). In some embodiments, a substitution in a coding sequence canbe nonsense mutation (e.g., a premature stop codon is encoded). It willbe understood that mutations can be used to alter an endogenous nucleicacid, using, for example, CRISPR, TALEN, and/or Zinc-finger nucleases.

In some embodiments, a mutation in a protein sequence can be aninsertion, a deletion, or a substitution. It will be understood that amutation in a nucleic acid that encodes a protein can cause a mutationin a protein sequence. In some embodiments, a mutation in a proteinsequence is a substitution (e.g., a cysteine to serine mutation, or acysteine to alanine mutation).

As used herein, a “corresponding” nucleic acid position (orsubstitution) in a nucleic acid sequence different from a referencenucleic acid sequence (e.g., in a truncated, extended, or mutatednucleic acid sequence of a pAOX1 promoter compared to a reference pAOXnucleic acid sequence, such as SEQ ID NO: 28) can be identified byperforming a sequence alignment between the nucleic acid sequences ofinterest. It will be understood that in some cases, a gap can exist in anucleic acid alignment. Similarly, a “corresponding” amino acid position(or substitution) in a protein sequence different from a referenceprotein sequence (e.g., in the myoglobin protein sequence of a differentorganism compared to a reference myoglobin protein sequence, such as SEQID NO: 18) can be identified by performing a sequence alignment betweenthe protein sequences of interest. It will be understood that in somecases, a gap can exist in a protein alignment. As used herein, anucleotide or amino acid position “relative to” a reference sequence canbe the corresponding nucleotide or amino acid position in a referencesequence.

In some embodiments, a reference sequence can be from the same taxonomicrank as a comparator sequence. In some embodiments, a reference sequencecan be from the same domain as a comparator sequence. For example, insome embodiments, both a reference sequence and a comparator sequencecan be from domain Eukarya. In some embodiments, a reference sequencecan be from the same kingdom as a comparator sequence. For example, insome embodiments, both a reference sequence and a comparator sequencecan be from the kingdom Fungi. In some embodiments, a reference sequencecan be from the same phylum as a comparator sequence. For example, insome embodiments, both a reference sequence and a comparator sequencecan be from phylum Ascomycota. In some embodiments, a reference sequencecan be from the same class as a comparator sequence. For example, insome embodiments, both a reference sequence and a comparator sequencecan be from the class Saccharomycetes. In some embodiments, a referencesequence can be from the same order as a comparator sequence. Forexample, in some embodiments, both a reference sequence and a comparatorsequence can be from the order Saccharomycetales. In some embodiments, areference sequence can be from the same family as a comparator sequence.For example, in some embodiments, both a reference sequence andcomparator sequence can be from the family Saccharomycetaceae. In someembodiments, a reference sequence can be from the same genus as acomparator sequence. For example, in some embodiments, both a referencesequence and a comparator sequence can be from the genus Pichia. In someembodiments, a reference sequence can be from the same species as acomparator sequence.

In some embodiments, a reference sequence and a comparator sequence canboth be from yeast. In some embodiments, a reference sequence and acomparator sequence can both be from methylotrophic yeast.

In some embodiments, a reference sequence and a comparator sequence canhave at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 99%) sequence identity.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: T146Cand C154T; T146C and T303C; T146C and T426A; T146C and A433T; T146C andA435G; T146C and T530A; T146C and C572T; T146C and T596C; T146C andT617C; T146C and T688C; T146C and A696T; T146C and T702C; T146C andA709G; T146C and A712G; T146C and T714G; T146C and A790G; T146C andA841T; T146C and T862A; C154T and T303C; C154T and T426A; C154T andA433T; C154T and A435G; C154T and T530A; C154T and C572T; C154T andT596C; C154T and T617C; C154T and T688C; C154T and A696T; C154T andT702C; C154T and A709G; C154T and A712G; C154T and T714G; C154T andA790G; C154T and A841T; C154T and T862A; T303C and T426A; T303C andA433T; T303C and A435G; T303C and T530A; T303C and C572T; T303C andT596C; T303C and T617C; T303C and T688C; T303C and A696T; T303C andT702C; T303C and A709G; T303C and A712G; T303C and T714G; T303C andA790G; T303C and A841T; T303C and T862A; T426A and A433T; T426A andA435G; T426A and T530A; T426A and C572T; T426A and T596C; T426A andT617C; T426A and T688C; T426A and A696T; T426A and T702C; T426A andA709G; T426A and A712G; T426A and T714G; T426A and A790G; T426A andA841T; T426A and T862A; A433T and A435G; A433T and T530A; A433T andC572T; A433T and T596C; A433T and T617C; A433T and T688C; A433T andA696T; A433T and T702C; A433T and A709G; A433T and A712G; A433T andT714G; A433T and A790G; A433T and A841T; A433T and T862A; A435G andT530A; A435G and C572T; A435G and T596C; A435G and T617C; A435G andT688C; A435G and A696T; A435G and T702C; A435G and A709G; A435G andA712G; A435G and T714G; A435G and A790G; A435G and A841T; A435G andT862A; T530A and C572T; T530A and T596C; T530A and T617C; T530A andT688C; T530A and A696T; T530A and T702C; T530A and A709G; T530A andA712G; T530A and T714G; T530A and A790G; T530A and A841T; T530A andT862A; C572T and T596C; C572T and T617C; C572T and T688C; C572T andA696T; C572T and T702C; C572T and A709G; C572T and A712G; C572T andT714G; C572T and A790G; C572T and A841T; C572T and T862A; T596C andT617C; T596C and T688C; T596C and A696T; T596C and T702C; T596C andA709G; T596C and A712G; T596C and T714G; T596C and A790G; T596C andA841T; T596C and T862A; T617C and T688C; T617C and A696T; T617C andT702C; T617C and A709G; T617C and A712G; T617C and T714G; T617C andA790G; T617C and A841T; T617C and T862A; T688C and A696T; T688C andT702C; T688C and A709G; T688C and A712G; T688C and T714G; T688C andA790G; T688C and A841T; T688C and T862A; A696T and T702C; A696T andA709G; A696T and A712G; A696T and T714G; A696T and A790G; A696T andA841T; A696T and T862A; T702C and A709G; T702C and A712G; T702C andT714G; T702C and A790G; T702C and A841T; T702C and T862A; A709G andA712G; A709G and T714G; A709G and A790G; A709G and A841T; A709G andT862A; A712G and T714G; A712G and A790G; A712G and A841T; A712G andT862A; T714G and A790G; T714G and A841T; T714G and T862A; A790G andA841T; A790G and T862A; or A841T and T862A.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations corresponding to the followingmutations relative to SEQ ID NO: 28 can be present in a promotersequence: T146C, C154T, and T303C; T146C, C154T, and T426A; T146C,C154T, and A433T; T146C, C154T, and A435G; T146C, C154T, and T530A;T146C, C154T, and C572T; T146C, C154T, and T596C; T146C, C154T, andT617C; T146C, C154T, and T688C; T146C, C154T, and A696T; T146C, C154T,and T702C; T146C, C154T, and A709G; T146C, C154T, and A712G; T146C,C154T, and T714G; T146C, C154T, and A790G; T146C, C154T, and A841T;T146C, C154T, and T862A; T146C, T303C, and T426A; T146C, T303C, andA433T; T146C, T303C, and A435G; T146C, T303C, and T530A; T146C, T303C,and C572T; T146C, T303C, and T596C; T146C, T303C, and T617C; T146C,T303C, and T688C; T146C, T303C, and A696T; T146C, T303C, and T702C;T146C, T303C, and A709G; T146C, T303C, and A712G; T146C, T303C, andT714G; T146C, T303C, and A790G; T146C, T303C, and A841T; T146C, T303C,and T862A; T146C, T426A, and A433T; T146C, T426A, and A435G; T146C,T426A, and T530A; T146C, T426A, and C572T; T146C, T426A, and T596C;T146C, T426A, and T617C; T146C, T426A, and T688C; T146C, T426A, andA696T; T146C, T426A, and T702C; T146C, T426A, and A709G; T146C, T426A,and A712G; T146C, T426A, and T714G; T146C, T426A, and A790G; T146C,T426A, and A841T; T146C, T426A, and T862A; T146C, A433T, and A435G;T146C, A433T, and T530A; T146C, A433T, and C572T; T146C, A433T, andT596C; T146C, A433T, and T617C; T146C, A433T, and T688C; T146C, A433T,and A696T; T146C, A433T, and T702C; T146C, A433T, and A709G; T146C,A433T, and A712G; T146C, A433T, and T714G; T146C, A433T, and A790G;T146C, A433T, and A841T; T146C, A433T, and T862A; T146C, A435G, andT530A; T146C, A435G, and C572T; T146C, A435G, and T596C; T146C, A435G,and T617C; T146C, A435G, and T688C; T146C, A435G, and A696T; T146C,A435G, and T702C; T146C, A435G, and A709G; T146C, A435G, and A712G;T146C, A435G, and T714G; T146C, A435G, and A790G; T146C, A435G, andA841T; T146C, A435G, and T862A; T146C, T530A, and C572T; T146C, T530A,and T596C; T146C, T530A, and T617C; T146C, T530A, and T688C; T146C,T530A, and A696T; T146C, T530A, and T702C; T146C, T530A, and A709G;T146C, T530A, and A712G; T146C, T530A, and T714G; T146C, T530A, andA790G; T146C, T530A, and A841T; T146C, T530A, and T862A; T146C, C572T,and T596C; T146C, C572T, and T617C; T146C, C572T, and T688C; T146C,C572T, and A696T; T146C, C572T, and T702C; T146C, C572T, and A709G;T146C, C572T, and A712G; T146C, C572T, and T714G; T146C, C572T, andA790G; T146C, C572T, and A841T; T146C, C572T, and T862A; T146C, T596C,and T617C; T146C, T596C, and T688C; T146C, T596C, and A696T; T146C,T596C, and T702C; T146C, T596C, and A709G; T146C, T596C, and A712G;T146C, T596C, and T714G; T146C, T596C, and A790G; T146C, T596C, andA841T; T146C, T596C, and T862A; T146C, T617C, and T688C; T146C, T617C,and A696T; T146C, T617C, and T702C; T146C, T617C, and A709G; T146C,T617C, and A712G; T146C, T617C, and T714G; T146C, T617C, and A790G;T146C, T617C, and A841T; T146C, T617C, and T862A; T146C, T688C, andA696T; T146C, T688C, and T702C; T146C, T688C, and A709G; T146C, T688C,and A712G; T146C, T688C, and T714G; T146C, T688C, and A790G; T146C,T688C, and A841T; T146C, T688C, and T862A; T146C, A696T, and T702C;T146C, A696T, and A709G; T146C, A696T, and A712G; T146C, A696T, andT714G; T146C, A696T, and A790G; T146C, A696T, and A841T; T146C, A696T,and T862A; T146C, T702C, and A709G; T146C, T702C, and A712G; T146C,T702C, and T714G; T146C, T702C, and A790G; T146C, T702C, and A841T;T146C, T702C, and T862A; T146C, A709G, and A712G; T146C, A709G, andT714G; T146C, A709G, and A790G; T146C, A709G, and A841T; T146C, A709G,and T862A; T146C, A712G, and T714G; T146C, A712G, and A790G; T146C,A712G, and A841T; T146C, A712G, and T862A; T146C, T714G, and A790G;T146C, T714G, and A841T; T146C, T714G, and T862A; T146C, A790G, andA841T; T146C, A790G, and T862A; T146C, A841T, and T862A; C154T, T303C,and T426A; C154T, T303C, and A433T; C154T, T303C, and A435G; C154T,T303C, and T530A; C154T, T303C, and C572T; C154T, T303C, and T596C;C154T, T303C, and T617C; C154T, T303C, and T688C; C154T, T303C, andA696T; C154T, T303C, and T702C; C154T, T303C, and A709G; C154T, T303C,and A712G; C154T, T303C, and T714G; C154T, T303C, and A790G; C154T,T303C, and A841T; C154T, T303C, and T862A; C154T, T426A, and A433T;C154T, T426A, and A435G; C154T, T426A, and T530A; C154T, T426A, andC572T; C154T, T426A, and T596C; C154T, T426A, and T617C; C154T, T426A,and T688C; C154T, T426A, and A696T; C154T, T426A, and T702C; C154T,T426A, and A709G; C154T, T426A, and A712G; C154T, T426A, and T714G;C154T, T426A, and A790G; C154T, T426A, and A841T; C154T, T426A, andT862A; C154T, A433T, and A435G; C154T, A433T, and T530A; C154T, A433T,and C572T; C154T, A433T, and T596C; C154T, A433T, and T617C; C154T,A433T, and T688C; C154T, A433T, and A696T; C154T, A433T, and T702C;C154T, A433T, and A709G; C154T, A433T, and A712G; C154T, A433T, andT714G; C154T, A433T, and A790G; C154T, A433T, and A841T; C154T, A433T,and T862A; C154T, A435G, and T530A; C154T, A435G, and C572T; C154T,A435G, and T596C; C154T, A435G, and T617C; C154T, A435G, and T688C;C154T, A435G, and A696T; C154T, A435G, and T702C; C154T, A435G, andA709G; C154T, A435G, and A712G; C154T, A435G, and T714G; C154T, A435G,and A790G; C154T, A435G, and A841T; C154T, A435G, and T862A; C154T,T530A, and C572T; C154T, T530A, and T596C; C154T, T530A, and T617C;C154T, T530A, and T688C; C154T, T530A, and A696T; C154T, T530A, andT702C; C154T, T530A, and A709G; C154T, T530A, and A712G; C154T, T530A,and T714G; C154T, T530A, and A790G; C154T, T530A, and A841T; C154T,T530A, and T862A; C154T, C572T, and T596C; C154T, C572T, and T617C;C154T, C572T, and T688C; C154T, C572T, and A696T; C154T, C572T, andT702C; C154T, C572T, and A709G; C154T, C572T, and A712G; C154T, C572T,and T714G; C154T, C572T, and A790G; C154T, C572T, and A841T; C154T,C572T, and T862A; C154T, T596C, and T617C; C154T, T596C, and T688C;C154T, T596C, and A696T; C154T, T596C, and T702C; C154T, T596C, andA709G; C154T, T596C, and A712G; C154T, T596C, and T714G; C154T, T596C,and A790G; C154T, T596C, and A841T; C154T, T596C, and T862A; C154T,T617C, and T688C; C154T, T617C, and A696T; C154T, T617C, and T702C;C154T, T617C, and A709G; C154T, T617C, and A712G; C154T, T617C, andT714G; C154T, T617C, and A790G; C154T, T617C, and A841T; C154T, T617C,and T862A; C154T, T688C, and A696T; C154T, T688C, and T702C; C154T,T688C, and A709G; C154T, T688C, and A712G; C154T, T688C, and T714G;C154T, T688C, and A790G; C154T, T688C, and A841T; C154T, T688C, andT862A; C154T, A696T, and T702C; C154T, A696T, and A709G; C154T, A696T,and A712G; C154T, A696T, and T714G; C154T, A696T, and A790G; C154T,A696T, and A841T; C154T, A696T, and T862A; C154T, T702C, and A709G;C154T, T702C, and A712G; C154T, T702C, and T714G; C154T, T702C, andA790G; C154T, T702C, and A841T; C154T, T702C, and T862A; C154T, A709G,and A712G; C154T, A709G, and T714G; C154T, A709G, and A790G; C154T,A709G, and A841T; C154T, A709G, and T862A; C154T, A712G, and T714G;C154T, A712G, and A790G; C154T, A712G, and A841T; C154T, A712G, andT862A; C154T, T714G, and A790G; C154T, T714G, and A841T; C154T, T714G,and T862A; C154T, A790G, and A841T; C154T, A790G, and T862A; C154T,A841T, and T862A; T303C, T426A, and A433T; T303C, T426A, and A435G;T303C, T426A, and T530A; T303C, T426A, and C572T; T303C, T426A, andT596C; T303C, T426A, and T617C; T303C, T426A, and T688C; T303C, T426A,and A696T; T303C, T426A, and T702C; T303C, T426A, and A709G; T303C,T426A, and A712G; T303C, T426A, and T714G; T303C, T426A, and A790G;T303C, T426A, and A841T; T303C, T426A, and T862A; T303C, A433T, andA435G; T303C, A433T, and T530A; T303C, A433T, and C572T; T303C, A433T,and T596C; T303C, A433T, and T617C; T303C, A433T, and T688C; T303C,A433T, and A696T; T303C, A433T, and T702C; T303C, A433T, and A709G;T303C, A433T, and A712G; T303C, A433T, and T714G; T303C, A433T, andA790G; T303C, A433T, and A841T; T303C, A433T, and T862A; T303C, A435G,and T530A; T303C, A435G, and C572T; T303C, A435G, and T596C; T303C,A435G, and T617C; T303C, A435G, and T688C; T303C, A435G, and A696T;T303C, A435G, and T702C; T303C, A435G, and A709G; T303C, A435G, andA712G; T303C, A435G, and T714G; T303C, A435G, and A790G; T303C, A435G,and A841T; T303C, A435G, and T862A; T303C, T530A, and C572T; T303C,T530A, and T596C; T303C, T530A, and T617C; T303C, T530A, and T688C;T303C, T530A, and A696T; T303C, T530A, and T702C; T303C, T530A, andA709G; T303C, T530A, and A712G; T303C, T530A, and T714G; T303C, T530A,and A790G; T303C, T530A, and A841T; T303C, T530A, and T862A; T303C,C572T, and T596C; T303C, C572T, and T617C; T303C, C572T, and T688C;T303C, C572T, and A696T; T303C, C572T, and T702C; T303C, C572T, andA709G; T303C, C572T, and A712G; T303C, C572T, and T714G; T303C, C572T,and A790G; T303C, C572T, and A841T; T303C, C572T, and T862A; T303C,T596C, and T617C; T303C, T596C, and T688C; T303C, T596C, and A696T;T303C, T596C, and T702C; T303C, T596C, and A709G; T303C, T596C, andA712G; T303C, T596C, and T714G; T303C, T596C, and A790G; T303C, T596C,and A841T; T303C, T596C, and T862A; T303C, T617C, and T688C; T303C,T617C, and A696T; T303C, T617C, and T702C; T303C, T617C, and A709G;T303C, T617C, and A712G; T303C, T617C, and T714G; T303C, T617C, andA790G; T303C, T617C, and A841T; T303C, T617C, and T862A; T303C, T688C,and A696T; T303C, T688C, and T702C; T303C, T688C, and A709G; T303C,T688C, and A712G; T303C, T688C, and T714G; T303C, T688C, and A790G;T303C, T688C, and A841T; T303C, T688C, and T862A; T303C, A696T, andT702C; T303C, A696T, and A709G; T303C, A696T, and A712G; T303C, A696T,and T714G; T303C, A696T, and A790G; T303C, A696T, and A841T; T303C,A696T, and T862A; T303C, T702C, and A709G; T303C, T702C, and A712G;T303C, T702C, and T714G; T303C, T702C, and A790G; T303C, T702C, andA841T; T303C, T702C, and T862A; T303C, A709G, and A712G; T303C, A709G,and T714G; T303C, A709G, and A790G; T303C, A709G, and A841T; T303C,A709G, and T862A; T303C, A712G, and T714G; T303C, A712G, and A790G;T303C, A712G, and A841T; T303C, A712G, and T862A; T303C, T714G, andA790G; T303C, T714G, and A841T; T303C, T714G, and T862A; T303C, A790G,and A841T; T303C, A790G, and T862A; T303C, A841T, and T862A; T426A,A433T, and A435G; T426A, A433T, and T530A; T426A, A433T, and C572T;T426A, A433T, and T596C; T426A, A433T, and T617C; T426A, A433T, andT688C; T426A, A433T, and A696T; T426A, A433T, and T702C; T426A, A433T,and A709G; T426A, A433T, and A712G; T426A, A433T, and T714G; T426A,A433T, and A790G; T426A, A433T, and A841T; T426A, A433T, and T862A;T426A, A435G, and T530A; T426A, A435G, and C572T; T426A, A435G, andT596C; T426A, A435G, and T617C; T426A, A435G, and T688C; T426A, A435G,and A696T; T426A, A435G, and T702C; T426A, A435G, and A709G; T426A,A435G, and A712G; T426A, A435G, and T714G; T426A, A435G, and A790G;T426A, A435G, and A841T; T426A, A435G, and T862A; T426A, T530A, andC572T; T426A, T530A, and T596C; T426A, T530A, and T617C; T426A, T530A,and T688C; T426A, T530A, and A696T; T426A, T530A, and T702C; T426A,T530A, and A709G; T426A, T530A, and A712G; T426A, T530A, and T714G;T426A, T530A, and A790G; T426A, T530A, and A841T; T426A, T530A, andT862A; T426A, C572T, and T596C; T426A, C572T, and T617C; T426A, C572T,and T688C; T426A, C572T, and A696T; T426A, C572T, and T702C; T426A,C572T, and A709G; T426A, C572T, and A712G; T426A, C572T, and T714G;T426A, C572T, and A790G; T426A, C572T, and A841T; T426A, C572T, andT862A; T426A, T596C, and T617C; T426A, T596C, and T688C; T426A, T596C,and A696T; T426A, T596C, and T702C; T426A, T596C, and A709G; T426A,T596C, and A712G; T426A, T596C, and T714G; T426A, T596C, and A790G;T426A, T596C, and A841T; T426A, T596C, and T862A; T426A, T617C, andT688C; T426A, T617C, and A696T; T426A, T617C, and T702C; T426A, T617C,and A709G; T426A, T617C, and A712G; T426A, T617C, and T714G; T426A,T617C, and A790G; T426A, T617C, and A841T; T426A, T617C, and T862A;T426A, T688C, and A696T; T426A, T688C, and T702C; T426A, T688C, andA709G; T426A, T688C, and A712G; T426A, T688C, and T714G; T426A, T688C,and A790G; T426A, T688C, and A841T; T426A, T688C, and T862A; T426A,A696T, and T702C; T426A, A696T, and A709G; T426A, A696T, and A712G;T426A, A696T, and T714G; T426A, A696T, and A790G; T426A, A696T, andA841T; T426A, A696T, and T862A; T426A, T702C, and A709G; T426A, T702C,and A712G; T426A, T702C, and T714G; T426A, T702C, and A790G; T426A,T702C, and A841T; T426A, T702C, and T862A; T426A, A709G, and A712G;T426A, A709G, and T714G; T426A, A709G, and A790G; T426A, A709G, andA841T; T426A, A709G, and T862A; T426A, A712G, and T714G; T426A, A712G,and A790G; T426A, A712G, and A841T; T426A, A712G, and T862A; T426A,T714G, and A790G; T426A, T714G, and A841T; T426A, T714G, and T862A;T426A, A790G, and A841T; T426A, A790G, and T862A; T426A, A841T, andT862A; A433T, A435G, and T530A; A433T, A435G, and C572T; A433T, A435G,and T596C; A433T, A435G, and T617C; A433T, A435G, and T688C; A433T,A435G, and A696T; A433T, A435G, and T702C; A433T, A435G, and A709G;A433T, A435G, and A712G; A433T, A435G, and T714G; A433T, A435G, andA790G; A433T, A435G, and A841T; A433T, A435G, and T862A; A433T, T530A,and C572T; A433T, T530A, and T596C; A433T, T530A, and T617C; A433T,T530A, and T688C; A433T, T530A, and A696T; A433T, T530A, and T702C;A433T, T530A, and A709G; A433T, T530A, and A712G; A433T, T530A, andT714G; A433T, T530A, and A790G; A433T, T530A, and A841T; A433T, T530A,and T862A; A433T, C572T, and T596C; A433T, C572T, and T617C; A433T,C572T, and T688C; A433T, C572T, and A696T; A433T, C572T, and T702C;A433T, C572T, and A709G; A433T, C572T, and A712G; A433T, C572T, andT714G; A433T, C572T, and A790G; A433T, C572T, and A841T; A433T, C572T,and T862A; A433T, T596C, and T617C; A433T, T596C, and T688C; A433T,T596C, and A696T; A433T, T596C, and T702C; A433T, T596C, and A709G;A433T, T596C, and A712G; A433T, T596C, and T714G; A433T, T596C, andA790G; A433T, T596C, and A841T; A433T, T596C, and T862A; A433T, T617C,and T688C; A433T, T617C, and A696T; A433T, T617C, and T702C; A433T,T617C, and A709G; A433T, T617C, and A712G; A433T, T617C, and T714G;A433T, T617C, and A790G; A433T, T617C, and A841T; A433T, T617C, andT862A; A433T, T688C, and A696T; A433T, T688C, and T702C; A433T, T688C,and A709G; A433T, T688C, and A712G; A433T, T688C, and T714G; A433T,T688C, and A790G; A433T, T688C, and A841T; A433T, T688C, and T862A;A433T, A696T, and T702C; A433T, A696T, and A709G; A433T, A696T, andA712G; A433T, A696T, and T714G; A433T, A696T, and A790G; A433T, A696T,and A841T; A433T, A696T, and T862A; A433T, T702C, and A709G; A433T,T702C, and A712G; A433T, T702C, and T714G; A433T, T702C, and A790G;A433T, T702C, and A841T; A433T, T702C, and T862A; A433T, A709G, andA712G; A433T, A709G, and T714G; A433T, A709G, and A790G; A433T, A709G,and A841T; A433T, A709G, and T862A; A433T, A712G, and T714G; A433T,A712G, and A790G; A433T, A712G, and A841T; A433T, A712G, and T862A;A433T, T714G, and A790G; A433T, T714G, and A841T; A433T, T714G, andT862A; A433T, A790G, and A841T; A433T, A790G, and T862A; A433T, A841T,and T862A; A435G, T530A, and C572T; A435G, T530A, and T596C; A435G,T530A, and T617C; A435G, T530A, and T688C; A435G, T530A, and A696T;A435G, T530A, and T702C; A435G, T530A, and A709G; A435G, T530A, andA712G; A435G, T530A, and T714G; A435G, T530A, and A790G; A435G, T530A,and A841T; A435G, T530A, and T862A; A435G, C572T, and T596C; A435G,C572T, and T617C; A435G, C572T, and T688C; A435G, C572T, and A696T;A435G, C572T, and T702C; A435G, C572T, and A709G; A435G, C572T, andA712G; A435G, C572T, and T714G; A435G, C572T, and A790G; A435G, C572T,and A841T; A435G, C572T, and T862A; A435G, T596C, and T617C; A435G,T596C, and T688C; A435G, T596C, and A696T; A435G, T596C, and T702C;A435G, T596C, and A709G; A435G, T596C, and A712G; A435G, T596C, andT714G; A435G, T596C, and A790G; A435G, T596C, and A841T; A435G, T596C,and T862A; A435G, T617C, and T688C; A435G, T617C, and A696T; A435G,T617C, and T702C; A435G, T617C, and A709G; A435G, T617C, and A712G;A435G, T617C, and T714G; A435G, T617C, and A790G; A435G, T617C, andA841T; A435G, T617C, and T862A; A435G, T688C, and A696T; A435G, T688C,and T702C; A435G, T688C, and A709G; A435G, T688C, and A712G; A435G,T688C, and T714G; A435G, T688C, and A790G; A435G, T688C, and A841T;A435G, T688C, and T862A; A435G, A696T, and T702C; A435G, A696T, andA709G; A435G, A696T, and A712G; A435G, A696T, and T714G; A435G, A696T,and A790G; A435G, A696T, and A841T; A435G, A696T, and T862A; A435G,T702C, and A709G; A435G, T702C, and A712G; A435G, T702C, and T714G;A435G, T702C, and A790G; A435G, T702C, and A841T; A435G, T702C, andT862A; A435G, A709G, and A712G; A435G, A709G, and T714G; A435G, A709G,and A790G; A435G, A709G, and A841T; A435G, A709G, and T862A; A435G,A712G, and T714G; A435G, A712G, and A790G; A435G, A712G, and A841T;A435G, A712G, and T862A; A435G, T714G, and A790G; A435G, T714G, andA841T; A435G, T714G, and T862A; A435G, A790G, and A841T; A435G, A790G,and T862A; A435G, A841T, and T862A; T530A, C572T, and T596C; T530A,C572T, and T617C; T530A, C572T, and T688C; T530A, C572T, and A696T;T530A, C572T, and T702C; T530A, C572T, and A709G; T530A, C572T, andA712G; T530A, C572T, and T714G; T530A, C572T, and A790G; T530A, C572T,and A841T; T530A, C572T, and T862A; T530A, T596C, and T617C; T530A,T596C, and T688C; T530A, T596C, and A696T; T530A, T596C, and T702C;T530A, T596C, and A709G; T530A, T596C, and A712G; T530A, T596C, andT714G; T530A, T596C, and A790G; T530A, T596C, and A841T; T530A, T596C,and T862A; T530A, T617C, and T688C; T530A, T617C, and A696T; T530A,T617C, and T702C; T530A, T617C, and A709G; T530A, T617C, and A712G;T530A, T617C, and T714G; T530A, T617C, and A790G; T530A, T617C, andA841T; T530A, T617C, and T862A; T530A, T688C, and A696T; T530A, T688C,and T702C; T530A, T688C, and A709G; T530A, T688C, and A712G; T530A,T688C, and T714G; T530A, T688C, and A790G; T530A, T688C, and A841T;T530A, T688C, and T862A; T530A, A696T, and T702C; T530A, A696T, andA709G; T530A, A696T, and A712G; T530A, A696T, and T714G; T530A, A696T,and A790G; T530A, A696T, and A841T; T530A, A696T, and T862A; T530A,T702C, and A709G; T530A, T702C, and A712G; T530A, T702C, and T714G;T530A, T702C, and A790G; T530A, T702C, and A841T; T530A, T702C, andT862A; T530A, A709G, and A712G; T530A, A709G, and T714G; T530A, A709G,and A790G; T530A, A709G, and A841T; T530A, A709G, and T862A; T530A,A712G, and T714G; T530A, A712G, and A790G; T530A, A712G, and A841T;T530A, A712G, and T862A; T530A, T714G, and A790G; T530A, T714G, andA841T; T530A, T714G, and T862A; T530A, A790G, and A841T; T530A, A790G,and T862A; T530A, A841T, and T862A; C572T, T596C, and T617C; C572T,T596C, and T688C; C572T, T596C, and A696T; C572T, T596C, and T702C;C572T, T596C, and A709G; C572T, T596C, and A712G; C572T, T596C, andT714G; C572T, T596C, and A790G; C572T, T596C, and A841T; C572T, T596C,and T862A; C572T, T617C, and T688C; C572T, T617C, and A696T; C572T,T617C, and T702C; C572T, T617C, and A709G; C572T, T617C, and A712G;C572T, T617C, and T714G; C572T, T617C, and A790G; C572T, T617C, andA841T; C572T, T617C, and T862A; C572T, T688C, and A696T; C572T, T688C,and T702C; C572T, T688C, and A709G; C572T, T688C, and A712G; C572T,T688C, and T714G; C572T, T688C, and A790G; C572T, T688C, and A841T;C572T, T688C, and T862A; C572T, A696T, and T702C; C572T, A696T, andA709G; C572T, A696T, and A712G; C572T, A696T, and T714G; C572T, A696T,and A790G; C572T, A696T, and A841T; C572T, A696T, and T862A; C572T,T702C, and A709G; C572T, T702C, and A712G; C572T, T702C, and T714G;C572T, T702C, and A790G; C572T, T702C, and A841T; C572T, T702C, andT862A; C572T, A709G, and A712G; C572T, A709G, and T714G; C572T, A709G,and A790G; C572T, A709G, and A841T; C572T, A709G, and T862A; C572T,A712G, and T714G; C572T, A712G, and A790G; C572T, A712G, and A841T;C572T, A712G, and T862A; C572T, T714G, and A790G; C572T, T714G, andA841T; C572T, T714G, and T862A; C572T, A790G, and A841T; C572T, A790G,and T862A; C572T, A841T, and T862A; T596C, T617C, and T688C; T596C,T617C, and A696T; T596C, T617C, and T702C; T596C, T617C, and A709G;T596C, T617C, and A712G; T596C, T617C, and T714G; T596C, T617C, andA790G; T596C, T617C, and A841T; T596C, T617C, and T862A; T596C, T688C,and A696T; T596C, T688C, and T702C; T596C, T688C, and A709G; T596C,T688C, and A712G; T596C, T688C, and T714G; T596C, T688C, and A790G;T596C, T688C, and A841T; T596C, T688C, and T862A; T596C, A696T, andT702C; T596C, A696T, and A709G; T596C, A696T, and A712G; T596C, A696T,and T714G; T596C, A696T, and A790G; T596C, A696T, and A841T; T596C,A696T, and T862A; T596C, T702C, and A709G; T596C, T702C, and A712G;T596C, T702C, and T714G; T596C, T702C, and A790G; T596C, T702C, andA841T; T596C, T702C, and T862A; T596C, A709G, and A712G; T596C, A709G,and T714G; T596C, A709G, and A790G; T596C, A709G, and A841T; T596C,A709G, and T862A; T596C, A712G, and T714G; T596C, A712G, and A790G;T596C, A712G, and A841T; T596C, A712G, and T862A; T596C, T714G, andA790G; T596C, T714G, and A841T; T596C, T714G, and T862A; T596C, A790G,and A841T; T596C, A790G, and T862A; T596C, A841T, and T862A; T617C,T688C, and A696T; T617C, T688C, and T702C; T617C, T688C, and A709G;T617C, T688C, and A712G; T617C, T688C, and T714G; T617C, T688C, andA790G; T617C, T688C, and A841T; T617C, T688C, and T862A; T617C, A696T,and T702C; T617C, A696T, and A709G; T617C, A696T, and A712G; T617C,A696T, and T714G; T617C, A696T, and A790G; T617C, A696T, and A841T;T617C, A696T, and T862A; T617C, T702C, and A709G; T617C, T702C, andA712G; T617C, T702C, and T714G; T617C, T702C, and A790G; T617C, T702C,and A841T; T617C, T702C, and T862A; T617C, A709G, and A712G; T617C,A709G, and T714G; T617C, A709G, and A790G; T617C, A709G, and A841T;T617C, A709G, and T862A; T617C, A712G, and T714G; T617C, A712G, andA790G; T617C, A712G, and A841T; T617C, A712G, and T862A; T617C, T714G,and A790G; T617C, T714G, and A841T; T617C, T714G, and T862A; T617C,A790G, and A841T; T617C, A790G, and T862A; T617C, A841T, and T862A;T688C, A696T, and T702C; T688C, A696T, and A709G; T688C, A696T, andA712G; T688C, A696T, and T714G; T688C, A696T, and A790G; T688C, A696T,and A841T; T688C, A696T, and T862A; T688C, T702C, and A709G; T688C,T702C, and A712G; T688C, T702C, and T714G; T688C, T702C, and A790G;T688C, T702C, and A841T; T688C, T702C, and T862A; T688C, A709G, andA712G; T688C, A709G, and T714G; T688C, A709G, and A790G; T688C, A709G,and A841T; T688C, A709G, and T862A; T688C, A712G, and T714G; T688C,A712G, and A790G; T688C, A712G, and A841T; T688C, A712G, and T862A;T688C, T714G, and A790G; T688C, T714G, and A841T; T688C, T714G, andT862A; T688C, A790G, and A841T; T688C, A790G, and T862A; T688C, A841T,and T862A; A696T, T702C, and A709G; A696T, T702C, and A712G; A696T,T702C, and T714G; A696T, T702C, and A790G; A696T, T702C, and A841T;A696T, T702C, and T862A; A696T, A709G, and A712G; A696T, A709G, andT714G; A696T, A709G, and A790G; A696T, A709G, and A841T; A696T, A709G,and T862A; A696T, A712G, and T714G; A696T, A712G, and A790G; A696T,A712G, and A841T; A696T, A712G, and T862A; A696T, T714G, and A790G;A696T, T714G, and A841T; A696T, T714G, and T862A; A696T, A790G, andA841T; A696T, A790G, and T862A; A696T, A841T, and T862A; T702C, A709G,and A712G; T702C, A709G, and T714G; T702C, A709G, and A790G; T702C,A709G, and A841T; T702C, A709G, and T862A; T702C, A712G, and T714G;T702C, A712G, and A790G; T702C, A712G, and A841T; T702C, A712G, andT862A; T702C, T714G, and A790G; T702C, T714G, and A841T; T702C, T714G,and T862A; T702C, A790G, and A841T; T702C, A790G, and T862A; T702C,A841T, and T862A; A709G, A712G, and T714G; A709G, A712G, and A790G;A709G, A712G, and A841T; A709G, A712G, and T862A; A709G, T714G, andA790G; A709G, T714G, and A841T; A709G, T714G, and T862A; A709G, A790G,and A841T; A709G, A790G, and T862A; A709G, A841T, and T862A; A712G,T714G, and A790G; A712G, T714G, and A841T; A712G, T714G, and T862A;A712G, A790G, and A841T; A712G, A790G, and T862A; A712G, A841T, andT862A; T714G, A790G, and A841T; T714G, A790G, and T862A; T714G, A841T,and T862A; or A790G, A841T, and T862A.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: T688Cand A696T; T688C and T702C; T688C and A712G; T688C and T714G; A696T andT702C; A696T and A712G; A696T and T714G; T702C and A712G; T702C andT714G; or A712G and T714G.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations corresponding to the followingmutations relative to SEQ ID NO: 28 can be present in a promotersequence: T688C, A696T, and T702C; T688C, A696T, and A712G; T688C,A696T, and T714G; T688C, T702C, and A712G; T688C, T702C, and T714G;T688C, A712G, and T714G; A696T, T702C, and A712G; A696T, T702C, andT714G; A696T, A712G, and T714G; or T702C, A712G, and T714G.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include four mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, four mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: T688C,A696T, T702C, and A712G; T688C, A696T, T702C, and T714G; T688C, A696T,A712G, and T714G; T688C, T702C, A712G, and T714G; or A696T, T702C,A712G, and T714G.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include five mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, five mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: T688C,A696T, T702C, A712G, and T714G.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: 146Cand 154T; 146C and 303C; 146C and 426A; 146C and 433T; 146C and 435G;146C and 530A; 146C and 572T; 146C and 596C; 146C and 617C; 146C and688C; 146C and 696T; 146C and 702C; 146C and 709G; 146C and 712G; 146Cand 714G; 146C and 790G; 146C and A841T; 146C and 862A; 154T and 303C;154T and 426A; 154T and 433T; 154T and 435G; 154T and 530A; 154T and572T; 154T and 596C; 154T and 617C; 154T and 688C; 154T and 696T; 154Tand 702C; 154T and 709G; 154T and 712G; 154T and 714G; 154T and 790G;154T and A841T; 154T and 862A; 303C and 426A; 303C and 433T; 303C and435G; 303C and 530A; 303C and 572T; 303C and 596C; 303C and 617C; 303Cand 688C; 303C and 696T; 303C and 702C; 303C and 709G; 303C and 712G;303C and 714G; 303C and 790G; 303C and A841T; 303C and 862A; 426A and433T; 426A and 435G; 426A and 530A; 426A and 572T; 426A and 596C; 426Aand 617C; 426A and 688C; 426A and 696T; 426A and 702C; 426A and 709G;426A and 712G; 426A and 714G; 426A and 790G; 426A and A841T; 426A and862A; 433T and 435G; 433T and 530A; 433T and 572T; 433T and 596C; 433Tand 617C; 433T and 688C; 433T and 696T; 433T and 702C; 433T and 709G;433T and 712G; 433T and 714G; 433T and 790G; 433T and A841T; 433T and862A; 435G and 530A; 435G and 572T; 435G and 596C; 435G and 617C; 435Gand 688C; 435G and 696T; 435G and 702C; 435G and 709G; 435G and 712G;435G and 714G; 435G and 790G; 435G and A841T; 435G and 862A; 530A and572T; 530A and 596C; 530A and 617C; 530A and 688C; 530A and 696T; 530Aand 702C; 530A and 709G; 530A and 712G; 530A and 714G; 530A and 790G;530A and A841T; 530A and 862A; 572T and 596C; 572T and 617C; 572T and688C; 572T and 696T; 572T and 702C; 572T and 709G; 572T and 712G; 572Tand 714G; 572T and 790G; 572T and A841T; 572T and 862A; 596C and 617C;596C and 688C; 596C and 696T; 596C and 702C; 596C and 709G; 596C and712G; 596C and 714G; 596C and 790G; 596C and A841T; 596C and 862A; 617Cand 688C; 617C and 696T; 617C and 702C; 617C and 709G; 617C and 712G;617C and 714G; 617C and 790G; 617C and A841T; 617C and 862A; 688C and696T; 688C and 702C; 688C and 709G; 688C and 712G; 688C and 714G; 688Cand 790G; 688C and A841T; 688C and 862A; 696T and 702C; 696T and 709G;696T and 712G; 696T and 714G; 696T and 790G; 696T and A841T; 696T and862A; 702C and 709G; 702C and 712G; 702C and 714G; 702C and 790G; 702Cand A841T; 702C and 862A; 709G and 712G; 709G and 714G; 709G and 790G;709G and A841T; 709G and 862A; 712G and 714G; 712G and 790G; 712G andA841T; 712G and 862A; 714G and 790G; 714G and A841T; 714G and 862A; 790Gand A841T; 790G and 862A; or A841T and 862A, as long as the indicatednucleobase is not the same as the corresponding naturally-occurringnucleobase.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations corresponding to the followingmutations relative to SEQ ID NO: 28 can be present in a promotersequence: 146C, 154T, and 303C; 146C, 154T, and 426A; 146C, 154T, and433T; 146C, 154T, and 435G; 146C, 154T, and 530A; 146C, 154T, and 572T;146C, 154T, and 596C; 146C, 154T, and 617C; 146C, 154T, and 688C; 146C,154T, and 696T; 146C, 154T, and 702C; 146C, 154T, and 709G; 146C, 154T,and 712G; 146C, 154T, and 714G; 146C, 154T, and 790G; 146C, 154T, andA841T; 146C, 154T, and 862A; 146C, 303C, and 426A; 146C, 303C, and 433T;146C, 303C, and 435G; 146C, 303C, and 530A; 146C, 303C, and 572T; 146C,303C, and 596C; 146C, 303C, and 617C; 146C, 303C, and 688C; 146C, 303C,and 696T; 146C, 303C, and 702C; 146C, 303C, and 709G; 146C, 303C, and712G; 146C, 303C, and 714G; 146C, 303C, and 790G; 146C, 303C, and A841T;146C, 303C, and 862A; 146C, 426A, and 433T; 146C, 426A, and 435G; 146C,426A, and 530A; 146C, 426A, and 572T; 146C, 426A, and 596C; 146C, 426A,and 617C; 146C, 426A, and 688C; 146C, 426A, and 696T; 146C, 426A, and702C; 146C, 426A, and 709G; 146C, 426A, and 712G; 146C, 426A, and 714G;146C, 426A, and 790G; 146C, 426A, and A841T; 146C, 426A, and 862A; 146C,433T, and 435G; 146C, 433T, and 530A; 146C, 433T, and 572T; 146C, 433T,and 596C; 146C, 433T, and 617C; 146C, 433T, and 688C; 146C, 433T, and696T; 146C, 433T, and 702C; 146C, 433T, and 709G; 146C, 433T, and 712G;146C, 433T, and 714G; 146C, 433T, and 790G; 146C, 433T, and A841T; 146C,433T, and 862A; 146C, 435G, and 530A; 146C, 435G, and 572T; 146C, 435G,and 596C; 146C, 435G, and 617C; 146C, 435G, and 688C; 146C, 435G, and696T; 146C, 435G, and 702C; 146C, 435G, and 709G; 146C, 435G, and 712G;146C, 435G, and 714G; 146C, 435G, and 790G; 146C, 435G, and A841T; 146C,435G, and 862A; 146C, 530A, and 572T; 146C, 530A, and 596C; 146C, 530A,and 617C; 146C, 530A, and 688C; 146C, 530A, and 696T; 146C, 530A, and702C; 146C, 530A, and 709G; 146C, 530A, and 712G; 146C, 530A, and 714G;146C, 530A, and 790G; 146C, 530A, and A841T; 146C, 530A, and 862A; 146C,572T, and 596C; 146C, 572T, and 617C; 146C, 572T, and 688C; 146C, 572T,and 696T; 146C, 572T, and 702C; 146C, 572T, and 709G; 146C, 572T, and712G; 146C, 572T, and 714G; 146C, 572T, and 790G; 146C, 572T, and A841T;146C, 572T, and 862A; 146C, 596C, and 617C; 146C, 596C, and 688C; 146C,596C, and 696T; 146C, 596C, and 702C; 146C, 596C, and 709G; 146C, 596C,and 712G; 146C, 596C, and 714G; 146C, 596C, and 790G; 146C, 596C, andA841T; 146C, 596C, and 862A; 146C, 617C, and 688C; 146C, 617C, and 696T;146C, 617C, and 702C; 146C, 617C, and 709G; 146C, 617C, and 712G; 146C,617C, and 714G; 146C, 617C, and 790G; 146C, 617C, and A841T; 146C, 617C,and 862A; 146C, 688C, and 696T; 146C, 688C, and 702C; 146C, 688C, and709G; 146C, 688C, and 712G; 146C, 688C, and 714G; 146C, 688C, and 790G;146C, 688C, and A841T; 146C, 688C, and 862A; 146C, 696T, and 702C; 146C,696T, and 709G; 146C, 696T, and 712G; 146C, 696T, and 714G; 146C, 696T,and 790G; 146C, 696T, and A841T; 146C, 696T, and 862A; 146C, 702C, and709G; 146C, 702C, and 712G; 146C, 702C, and 714G; 146C, 702C, and 790G;146C, 702C, and A841T; 146C, 702C, and 862A; 146C, 709G, and 712G; 146C,709G, and 714G; 146C, 709G, and 790G; 146C, 709G, and A841T; 146C, 709G,and 862A; 146C, 712G, and 714G; 146C, 712G, and 790G; 146C, 712G, andA841T; 146C, 712G, and 862A; 146C, 714G, and 790G; 146C, 714G, andA841T; 146C, 714G, and 862A; 146C, 790G, and A841T; 146C, 790G, and862A; 146C, A841T, and 862A; 154T, 303C, and 426A; 154T, 303C, and 433T;154T, 303C, and 435G; 154T, 303C, and 530A; 154T, 303C, and 572T; 154T,303C, and 596C; 154T, 303C, and 617C; 154T, 303C, and 688C; 154T, 303C,and 696T; 154T, 303C, and 702C; 154T, 303C, and 709G; 154T, 303C, and712G; 154T, 303C, and 714G; 154T, 303C, and 790G; 154T, 303C, and A841T;154T, 303C, and 862A; 154T, 426A, and 433T; 154T, 426A, and 435G; 154T,426A, and 530A; 154T, 426A, and 572T; 154T, 426A, and 596C; 154T, 426A,and 617C; 154T, 426A, and 688C; 154T, 426A, and 696T; 154T, 426A, and702C; 154T, 426A, and 709G; 154T, 426A, and 712G; 154T, 426A, and 714G;154T, 426A, and 790G; 154T, 426A, and A841T; 154T, 426A, and 862A; 154T,433T, and 435G; 154T, 433T, and 530A; 154T, 433T, and 572T; 154T, 433T,and 596C; 154T, 433T, and 617C; 154T, 433T, and 688C; 154T, 433T, and696T; 154T, 433T, and 702C; 154T, 433T, and 709G; 154T, 433T, and 712G;154T, 433T, and 714G; 154T, 433T, and 790G; 154T, 433T, and A841T; 154T,433T, and 862A; 154T, 435G, and 530A; 154T, 435G, and 572T; 154T, 435G,and 596C; 154T, 435G, and 617C; 154T, 435G, and 688C; 154T, 435G, and696T; 154T, 435G, and 702C; 154T, 435G, and 709G; 154T, 435G, and 712G;154T, 435G, and 714G; 154T, 435G, and 790G; 154T, 435G, and A841T; 154T,435G, and 862A; 154T, 530A, and 572T; 154T, 530A, and 596C; 154T, 530A,and 617C; 154T, 530A, and 688C; 154T, 530A, and 696T; 154T, 530A, and702C; 154T, 530A, and 709G; 154T, 530A, and 712G; 154T, 530A, and 714G;154T, 530A, and 790G; 154T, 530A, and A841T; 154T, 530A, and 862A; 154T,572T, and 596C; 154T, 572T, and 617C; 154T, 572T, and 688C; 154T, 572T,and 696T; 154T, 572T, and 702C; 154T, 572T, and 709G; 154T, 572T, and712G; 154T, 572T, and 714G; 154T, 572T, and 790G; 154T, 572T, and A841T;154T, 572T, and 862A; 154T, 596C, and 617C; 154T, 596C, and 688C; 154T,596C, and 696T; 154T, 596C, and 702C; 154T, 596C, and 709G; 154T, 596C,and 712G; 154T, 596C, and 714G; 154T, 596C, and 790G; 154T, 596C, andA841T; 154T, 596C, and 862A; 154T, 617C, and 688C; 154T, 617C, and 696T;154T, 617C, and 702C; 154T, 617C, and 709G; 154T, 617C, and 712G; 154T,617C, and 714G; 154T, 617C, and 790G; 154T, 617C, and A841T; 154T, 617C,and 862A; 154T, 688C, and 696T; 154T, 688C, and 702C; 154T, 688C, and709G; 154T, 688C, and 712G; 154T, 688C, and 714G; 154T, 688C, and 790G;154T, 688C, and A841T; 154T, 688C, and 862A; 154T, 696T, and 702C; 154T,696T, and 709G; 154T, 696T, and 712G; 154T, 696T, and 714G; 154T, 696T,and 790G; 154T, 696T, and A841T; 154T, 696T, and 862A; 154T, 702C, and709G; 154T, 702C, and 712G; 154T, 702C, and 714G; 154T, 702C, and 790G;154T, 702C, and A841T; 154T, 702C, and 862A; 154T, 709G, and 712G; 154T,709G, and 714G; 154T, 709G, and 790G; 154T, 709G, and A841T; 154T, 709G,and 862A; 154T, 712G, and 714G; 154T, 712G, and 790G; 154T, 712G, andA841T; 154T, 712G, and 862A; 154T, 714G, and 790G; 154T, 714G, andA841T; 154T, 714G, and 862A; 154T, 790G, and A841T; 154T, 790G, and862A; 154T, A841T, and 862A; 303C, 426A, and 433T; 303C, 426A, and 435G;303C, 426A, and 530A; 303C, 426A, and 572T; 303C, 426A, and 596C; 303C,426A, and 617C; 303C, 426A, and 688C; 303C, 426A, and 696T; 303C, 426A,and 702C; 303C, 426A, and 709G; 303C, 426A, and 712G; 303C, 426A, and714G; 303C, 426A, and 790G; 303C, 426A, and A841T; 303C, 426A, and 862A;303C, 433T, and 435G; 303C, 433T, and 530A; 303C, 433T, and 572T; 303C,433T, and 596C; 303C, 433T, and 617C; 303C, 433T, and 688C; 303C, 433T,and 696T; 303C, 433T, and 702C; 303C, 433T, and 709G; 303C, 433T, and712G; 303C, 433T, and 714G; 303C, 433T, and 790G; 303C, 433T, and A841T;303C, 433T, and 862A; 303C, 435G, and 530A; 303C, 435G, and 572T; 303C,435G, and 596C; 303C, 435G, and 617C; 303C, 435G, and 688C; 303C, 435G,and 696T; 303C, 435G, and 702C; 303C, 435G, and 709G; 303C, 435G, and712G; 303C, 435G, and 714G; 303C, 435G, and 790G; 303C, 435G, and A841T;303C, 435G, and 862A; 303C, 530A, and 572T; 303C, 530A, and 596C; 303C,530A, and 617C; 303C, 530A, and 688C; 303C, 530A, and 696T; 303C, 530A,and 702C; 303C, 530A, and 709G; 303C, 530A, and 712G; 303C, 530A, and714G; 303C, 530A, and 790G; 303C, 530A, and A841T; 303C, 530A, and 862A;303C, 572T, and 596C; 303C, 572T, and 617C; 303C, 572T, and 688C; 303C,572T, and 696T; 303C, 572T, and 702C; 303C, 572T, and 709G; 303C, 572T,and 712G; 303C, 572T, and 714G; 303C, 572T, and 790G; 303C, 572T, andA841T; 303C, 572T, and 862A; 303C, 596C, and 617C; 303C, 596C, and 688C;303C, 596C, and 696T; 303C, 596C, and 702C; 303C, 596C, and 709G; 303C,596C, and 712G; 303C, 596C, and 714G; 303C, 596C, and 790G; 303C, 596C,and A841T; 303C, 596C, and 862A; 303C, 617C, and 688C; 303C, 617C, and696T; 303C, 617C, and 702C; 303C, 617C, and 709G; 303C, 617C, and 712G;303C, 617C, and 714G; 303C, 617C, and 790G; 303C, 617C, and A841T; 303C,617C, and 862A; 303C, 688C, and 696T; 303C, 688C, and 702C; 303C, 688C,and 709G; 303C, 688C, and 712G; 303C, 688C, and 714G; 303C, 688C, and790G; 303C, 688C, and A841T; 303C, 688C, and 862A; 303C, 696T, and 702C;303C, 696T, and 709G; 303C, 696T, and 712G; 303C, 696T, and 714G; 303C,696T, and 790G; 303C, 696T, and A841T; 303C, 696T, and 862A; 303C, 702C,and 709G; 303C, 702C, and 712G; 303C, 702C, and 714G; 303C, 702C, and790G; 303C, 702C, and A841T; 303C, 702C, and 862A; 303C, 709G, and 712G;303C, 709G, and 714G; 303C, 709G, and 790G; 303C, 709G, and A841T; 303C,709G, and 862A; 303C, 712G, and 714G; 303C, 712G, and 790G; 303C, 712G,and A841T; 303C, 712G, and 862A; 303C, 714G, and 790G; 303C, 714G, andA841T; 303C, 714G, and 862A; 303C, 790G, and A841T; 303C, 790G, and862A; 303C, A841T, and 862A; 426A, 433T, and 435G; 426A, 433T, and 530A;426A, 433T, and 572T; 426A, 433T, and 596C; 426A, 433T, and 617C; 426A,433T, and 688C; 426A, 433T, and 696T; 426A, 433T, and 702C; 426A, 433T,and 709G; 426A, 433T, and 712G; 426A, 433T, and 714G; 426A, 433T, and790G; 426A, 433T, and A841T; 426A, 433T, and 862A; 426A, 435G, and 530A;426A, 435G, and 572T; 426A, 435G, and 596C; 426A, 435G, and 617C; 426A,435G, and 688C; 426A, 435G, and 696T; 426A, 435G, and 702C; 426A, 435G,and 709G; 426A, 435G, and 712G; 426A, 435G, and 714G; 426A, 435G, and790G; 426A, 435G, and A841T; 426A, 435G, and 862A; 426A, 530A, and 572T;426A, 530A, and 596C; 426A, 530A, and 617C; 426A, 530A, and 688C; 426A,530A, and 696T; 426A, 530A, and 702C; 426A, 530A, and 709G; 426A, 530A,and 712G; 426A, 530A, and 714G; 426A, 530A, and 790G; 426A, 530A, andA841T; 426A, 530A, and 862A; 426A, 572T, and 596C; 426A, 572T, and 617C;426A, 572T, and 688C; 426A, 572T, and 696T; 426A, 572T, and 702C; 426A,572T, and 709G; 426A, 572T, and 712G; 426A, 572T, and 714G; 426A, 572T,and 790G; 426A, 572T, and A841T; 426A, 572T, and 862A; 426A, 596C, and617C; 426A, 596C, and 688C; 426A, 596C, and 696T; 426A, 596C, and 702C;426A, 596C, and 709G; 426A, 596C, and 712G; 426A, 596C, and 714G; 426A,596C, and 790G; 426A, 596C, and A841T; 426A, 596C, and 862A; 426A, 617C,and 688C; 426A, 617C, and 696T; 426A, 617C, and 702C; 426A, 617C, and709G; 426A, 617C, and 712G; 426A, 617C, and 714G; 426A, 617C, and 790G;426A, 617C, and A841T; 426A, 617C, and 862A; 426A, 688C, and 696T; 426A,688C, and 702C; 426A, 688C, and 709G; 426A, 688C, and 712G; 426A, 688C,and 714G; 426A, 688C, and 790G; 426A, 688C, and A841T; 426A, 688C, and862A; 426A, 696T, and 702C; 426A, 696T, and 709G; 426A, 696T, and 712G;426A, 696T, and 714G; 426A, 696T, and 790G; 426A, 696T, and A841T; 426A,696T, and 862A; 426A, 702C, and 709G; 426A, 702C, and 712G; 426A, 702C,and 714G; 426A, 702C, and 790G; 426A, 702C, and A841T; 426A, 702C, and862A; 426A, 709G, and 712G; 426A, 709G, and 714G; 426A, 709G, and 790G;426A, 709G, and A841T; 426A, 709G, and 862A; 426A, 712G, and 714G; 426A,712G, and 790G; 426A, 712G, and A841T; 426A, 712G, and 862A; 426A, 714G,and 790G; 426A, 714G, and A841T; 426A, 714G, and 862A; 426A, 790G, andA841T; 426A, 790G, and 862A; 426A, A841T, and 862A; 433T, 435G, and530A; 433T, 435G, and 572T; 433T, 435G, and 596C; 433T, 435G, and 617C;433T, 435G, and 688C; 433T, 435G, and 696T; 433T, 435G, and 702C; 433T,435G, and 709G; 433T, 435G, and 712G; 433T, 435G, and 714G; 433T, 435G,and 790G; 433T, 435G, and A841T; 433T, 435G, and 862A; 433T, 530A, and572T; 433T, 530A, and 596C; 433T, 530A, and 617C; 433T, 530A, and 688C;433T, 530A, and 696T; 433T, 530A, and 702C; 433T, 530A, and 709G; 433T,530A, and 712G; 433T, 530A, and 714G; 433T, 530A, and 790G; 433T, 530A,and A841T; 433T, 530A, and 862A; 433T, 572T, and 596C; 433T, 572T, and617C; 433T, 572T, and 688C; 433T, 572T, and 696T; 433T, 572T, and 702C;433T, 572T, and 709G; 433T, 572T, and 712G; 433T, 572T, and 714G; 433T,572T, and 790G; 433T, 572T, and A841T; 433T, 572T, and 862A; 433T, 596C,and 617C; 433T, 596C, and 688C; 433T, 596C, and 696T; 433T, 596C, and702C; 433T, 596C, and 709G; 433T, 596C, and 712G; 433T, 596C, and 714G;433T, 596C, and 790G; 433T, 596C, and A841T; 433T, 596C, and 862A; 433T,617C, and 688C; 433T, 617C, and 696T; 433T, 617C, and 702C; 433T, 617C,and 709G; 433T, 617C, and 712G; 433T, 617C, and 714G; 433T, 617C, and790G; 433T, 617C, and A841T; 433T, 617C, and 862A; 433T, 688C, and 696T;433T, 688C, and 702C; 433T, 688C, and 709G; 433T, 688C, and 712G; 433T,688C, and 714G; 433T, 688C, and 790G; 433T, 688C, and A841T; 433T, 688C,and 862A; 433T, 696T, and 702C; 433T, 696T, and 709G; 433T, 696T, and712G; 433T, 696T, and 714G; 433T, 696T, and 790G; 433T, 696T, and A841T;433T, 696T, and 862A; 433T, 702C, and 709G; 433T, 702C, and 712G; 433T,702C, and 714G; 433T, 702C, and 790G; 433T, 702C, and A841T; 433T, 702C,and 862A; 433T, 709G, and 712G; 433T, 709G, and 714G; 433T, 709G, and790G; 433T, 709G, and A841T; 433T, 709G, and 862A; 433T, 712G, and 714G;433T, 712G, and 790G; 433T, 712G, and A841T; 433T, 712G, and 862A; 433T,714G, and 790G; 433T, 714G, and A841T; 433T, 714G, and 862A; 433T, 790G,and A841T; 433T, 790G, and 862A; 433T, A841T, and 862A; 435G, 530A, and572T; 435G, 530A, and 596C; 435G, 530A, and 617C; 435G, 530A, and 688C;435G, 530A, and 696T; 435G, 530A, and 702C; 435G, 530A, and 709G; 435G,530A, and 712G; 435G, 530A, and 714G; 435G, 530A, and 790G; 435G, 530A,and A841T; 435G, 530A, and 862A; 435G, 572T, and 596C; 435G, 572T, and617C; 435G, 572T, and 688C; 435G, 572T, and 696T; 435G, 572T, and 702C;435G, 572T, and 709G; 435G, 572T, and 712G; 435G, 572T, and 714G; 435G,572T, and 790G; 435G, 572T, and A841T; 435G, 572T, and 862A; 435G, 596C,and 617C; 435G, 596C, and 688C; 435G, 596C, and 696T; 435G, 596C, and702C; 435G, 596C, and 709G; 435G, 596C, and 712G; 435G, 596C, and 714G;435G, 596C, and 790G; 435G, 596C, and A841T; 435G, 596C, and 862A; 435G,617C, and 688C; 435G, 617C, and 696T; 435G, 617C, and 702C; 435G, 617C,and 709G; 435G, 617C, and 712G; 435G, 617C, and 714G; 435G, 617C, and790G; 435G, 617C, and A841T; 435G, 617C, and 862A; 435G, 688C, and 696T;435G, 688C, and 702C; 435G, 688C, and 709G; 435G, 688C, and 712G; 435G,688C, and 714G; 435G, 688C, and 790G; 435G, 688C, and A841T; 435G, 688C,and 862A; 435G, 696T, and 702C; 435G, 696T, and 709G; 435G, 696T, and712G; 435G, 696T, and 714G; 435G, 696T, and 790G; 435G, 696T, and A841T;435G, 696T, and 862A; 435G, 702C, and 709G; 435G, 702C, and 712G; 435G,702C, and 714G; 435G, 702C, and 790G; 435G, 702C, and A841T; 435G, 702C,and 862A; 435G, 709G, and 712G; 435G, 709G, and 714G; 435G, 709G, and790G; 435G, 709G, and A841T; 435G, 709G, and 862A; 435G, 712G, and 714G;435G, 712G, and 790G; 435G, 712G, and A841T; 435G, 712G, and 862A; 435G,714G, and 790G; 435G, 714G, and A841T; 435G, 714G, and 862A; 435G, 790G,and A841T; 435G, 790G, and 862A; 435G, A841T, and 862A; 530A, 572T, and596C; 530A, 572T, and 617C; 530A, 572T, and 688C; 530A, 572T, and 696T;530A, 572T, and 702C; 530A, 572T, and 709G; 530A, 572T, and 712G; 530A,572T, and 714G; 530A, 572T, and 790G; 530A, 572T, and A841T; 530A, 572T,and 862A; 530A, 596C, and 617C; 530A, 596C, and 688C; 530A, 596C, and696T; 530A, 596C, and 702C; 530A, 596C, and 709G; 530A, 596C, and 712G;530A, 596C, and 714G; 530A, 596C, and 790G; 530A, 596C, and A841T; 530A,596C, and 862A; 530A, 617C, and 688C; 530A, 617C, and 696T; 530A, 617C,and 702C; 530A, 617C, and 709G; 530A, 617C, and 712G; 530A, 617C, and714G; 530A, 617C, and 790G; 530A, 617C, and A841T; 530A, 617C, and 862A;530A, 688C, and 696T; 530A, 688C, and 702C; 530A, 688C, and 709G; 530A,688C, and 712G; 530A, 688C, and 714G; 530A, 688C, and 790G; 530A, 688C,and A841T; 530A, 688C, and 862A; 530A, 696T, and 702C; 530A, 696T, and709G; 530A, 696T, and 712G; 530A, 696T, and 714G; 530A, 696T, and 790G;530A, 696T, and A841T; 530A, 696T, and 862A; 530A, 702C, and 709G; 530A,702C, and 712G; 530A, 702C, and 714G; 530A, 702C, and 790G; 530A, 702C,and A841T; 530A, 702C, and 862A; 530A, 709G, and 712G; 530A, 709G, and714G; 530A, 709G, and 790G; 530A, 709G, and A841T; 530A, 709G, and 862A;530A, 712G, and 714G; 530A, 712G, and 790G; 530A, 712G, and A841T; 530A,712G, and 862A; 530A, 714G, and 790G; 530A, 714G, and A841T; 530A, 714G,and 862A; 530A, 790G, and A841T; 530A, 790G, and 862A; 530A, A841T, and862A; 572T, 596C, and 617C; 572T, 596C, and 688C; 572T, 596C, and 696T;572T, 596C, and 702C; 572T, 596C, and 709G; 572T, 596C, and 712G; 572T,596C, and 714G; 572T, 596C, and 790G; 572T, 596C, and A841T; 572T, 596C,and 862A; 572T, 617C, and 688C; 572T, 617C, and 696T; 572T, 617C, and702C; 572T, 617C, and 709G; 572T, 617C, and 712G; 572T, 617C, and 714G;572T, 617C, and 790G; 572T, 617C, and A841T; 572T, 617C, and 862A; 572T,688C, and 696T; 572T, 688C, and 702C; 572T, 688C, and 709G; 572T, 688C,and 712G; 572T, 688C, and 714G; 572T, 688C, and 790G; 572T, 688C, andA841T; 572T, 688C, and 862A; 572T, 696T, and 702C; 572T, 696T, and 709G;572T, 696T, and 712G; 572T, 696T, and 714G; 572T, 696T, and 790G; 572T,696T, and A841T; 572T, 696T, and 862A; 572T, 702C, and 709G; 572T, 702C,and 712G; 572T, 702C, and 714G; 572T, 702C, and 790G; 572T, 702C, andA841T; 572T, 702C, and 862A; 572T, 709G, and 712G; 572T, 709G, and 714G;572T, 709G, and 790G; 572T, 709G, and A841T; 572T, 709G, and 862A; 572T,712G, and 714G; 572T, 712G, and 790G; 572T, 712G, and A841T; 572T, 712G,and 862A; 572T, 714G, and 790G; 572T, 714G, and A841T; 572T, 714G, and862A; 572T, 790G, and A841T; 572T, 790G, and 862A; 572T, A841T, and862A; 596C, 617C, and 688C; 596C, 617C, and 696T; 596C, 617C, and 702C;596C, 617C, and 709G; 596C, 617C, and 712G; 596C, 617C, and 714G; 596C,617C, and 790G; 596C, 617C, and A841T; 596C, 617C, and 862A; 596C, 688C,and 696T; 596C, 688C, and 702C; 596C, 688C, and 709G; 596C, 688C, and712G; 596C, 688C, and 714G; 596C, 688C, and 790G; 596C, 688C, and A841T;596C, 688C, and 862A; 596C, 696T, and 702C; 596C, 696T, and 709G; 596C,696T, and 712G; 596C, 696T, and 714G; 596C, 696T, and 790G; 596C, 696T,and A841T; 596C, 696T, and 862A; 596C, 702C, and 709G; 596C, 702C, and712G; 596C, 702C, and 714G; 596C, 702C, and 790G; 596C, 702C, and A841T;596C, 702C, and 862A; 596C, 709G, and 712G; 596C, 709G, and 714G; 596C,709G, and 790G; 596C, 709G, and A841T; 596C, 709G, and 862A; 596C, 712G,and 714G; 596C, 712G, and 790G; 596C, 712G, and A841T; 596C, 712G, and862A; 596C, 714G, and 790G; 596C, 714G, and A841T; 596C, 714G, and 862A;596C, 790G, and A841T; 596C, 790G, and 862A; 596C, A841T, and 862A;617C, 688C, and 696T; 617C, 688C, and 702C; 617C, 688C, and 709G; 617C,688C, and 712G; 617C, 688C, and 714G; 617C, 688C, and 790G; 617C, 688C,and A841T; 617C, 688C, and 862A; 617C, 696T, and 702C; 617C, 696T, and709G; 617C, 696T, and 712G; 617C, 696T, and 714G; 617C, 696T, and 790G;617C, 696T, and A841T; 617C, 696T, and 862A; 617C, 702C, and 709G; 617C,702C, and 712G; 617C, 702C, and 714G; 617C, 702C, and 790G; 617C, 702C,and A841T; 617C, 702C, and 862A; 617C, 709G, and 712G; 617C, 709G, and714G; 617C, 709G, and 790G; 617C, 709G, and A841T; 617C, 709G, and 862A;617C, 712G, and 714G; 617C, 712G, and 790G; 617C, 712G, and A841T; 617C,712G, and 862A; 617C, 714G, and 790G; 617C, 714G, and A841T; 617C, 714G,and 862A; 617C, 790G, and A841T; 617C, 790G, and 862A; 617C, A841T, and862A; 688C, 696T, and 702C; 688C, 696T, and 709G; 688C, 696T, and 712G;688C, 696T, and 714G; 688C, 696T, and 790G; 688C, 696T, and A841T; 688C,696T, and 862A; 688C, 702C, and 709G; 688C, 702C, and 712G; 688C, 702C,and 714G; 688C, 702C, and 790G; 688C, 702C, and A841T; 688C, 702C, and862A; 688C, 709G, and 712G; 688C, 709G, and 714G; 688C, 709G, and 790G;688C, 709G, and A841T; 688C, 709G, and 862A; 688C, 712G, and 714G; 688C,712G, and 790G; 688C, 712G, and A841T; 688C, 712G, and 862A; 688C, 714G,and 790G; 688C, 714G, and A841T; 688C, 714G, and 862A; 688C, 790G, andA841T; 688C, 790G, and 862A; 688C, A841T, and 862A; 696T, 702C, and709G; 696T, 702C, and 712G; 696T, 702C, and 714G; 696T, 702C, and 790G;696T, 702C, and A841T; 696T, 702C, and 862A; 696T, 709G, and 712G; 696T,709G, and 714G; 696T, 709G, and 790G; 696T, 709G, and A841T; 696T, 709G,and 862A; 696T, 712G, and 714G; 696T, 712G, and 790G; 696T, 712G, andA841T; 696T, 712G, and 862A; 696T, 714G, and 790G; 696T, 714G, andA841T; 696T, 714G, and 862A; 696T, 790G, and A841T; 696T, 790G, and862A; 696T, A841T, and 862A; 702C, 709G, and 712G; 702C, 709G, and 714G;702C, 709G, and 790G; 702C, 709G, and A841T; 702C, 709G, and 862A; 702C,712G, and 714G; 702C, 712G, and 790G; 702C, 712G, and A841T; 702C, 712G,and 862A; 702C, 714G, and 790G; 702C, 714G, and A841T; 702C, 714G, and862A; 702C, 790G, and A841T; 702C, 790G, and 862A; 702C, A841T, and862A; 709G, 712G, and 714G; 709G, 712G, and 790G; 709G, 712G, and A841T;709G, 712G, and 862A; 709G, 714G, and 790G; 709G, 714G, and A841T; 709G,714G, and 862A; 709G, 790G, and A841T; 709G, 790G, and 862A; 709G,A841T, and 862A; 712G, 714G, and 790G; 712G, 714G, and A841T; 712G,714G, and 862A; 712G, 790G, and A841T; 712G, 790G, and 862A; 712G,A841T, and 862A; 714G, 790G, and A841T; 714G, 790G, and 862A; 714G,A841T, and 862A; or 790G, A841T, and 862A, as long as the indicatednucleobase is not the same as the corresponding naturally-occurringnucleobase.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: 688Cand 696T; 688C and 702C; 688C and 712G; 688C and 714G; 696T and 702C;696T and 712G; 696T and 714G; 702C and 712G; 702C and 714G; or 712G and714G, as long as the indicated nucleobase is not the same as thecorresponding naturally-occurring nucleobase.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations corresponding to the followingmutations relative to SEQ ID NO: 28 can be present in a promotersequence: 688C, 696T, and 702C; 688C, 696T, and 712G; 688C, 696T, and714G; 688C, 702C, and 712G; 688C, 702C, and 714G; 688C, 712G, and 714G;696T, 702C, and 712G; 696T, 702C, and 714G; 696T, 712G, and 714G; or702C, 712G, and 714G, as long as the indicated nucleobase is not thesame as the corresponding naturally-occurring nucleobase.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include four mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, four mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: 688C,696T, 702C, and 712G; 688C, 696T, 702C, and 714G; 688C, 696T, 712G, and714G; 688C, 702C, 712G, and 714G; or 696T, 702C, 712G, and 714G, as longas the indicated nucleobase is not the same as the correspondingnaturally-occurring nucleobase.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include five mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, five mutations corresponding to the following mutationsrelative to SEQ ID NO: 28 can be present in a promoter sequence: 688C,696T, 702C, 712G, and 714G, as long as the indicated nucleobase is notthe same as the corresponding naturally-occurring nucleobase.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: T146 and C154; T146 and T303; T146 and T426; T146 and A433;T146 and A435; T146 and T530; T146 and C572; T146 and T596; T146 andT617; T146 and T688; T146 and A696; T146 and T702; T146 and A709; T146and A712; T146 and T714; T146 and A790; T146 and A841; T146 and T862;C154 and T303; C154 and T426; C154 and A433; C154 and A435; C154 andT530; C154 and C572; C154 and T596; C154 and T617; C154 and T688; C154and A696; C154 and T702; C154 and A709; C154 and A712; C154 and T714;C154 and A790; C154 and A841; C154 and T862; T303 and T426; T303 andA433; T303 and A435; T303 and T530; T303 and C572; T303 and T596; T303and T617; T303 and T688; T303 and A696; T303 and T702; T303 and A709;T303 and A712; T303 and T714; T303 and A790; T303 and A841; T303 andT862; T426 and A433; T426 and A435; T426 and T530; T426 and C572; T426and T596; T426 and T617; T426 and T688; T426 and A696; T426 and T702;T426 and A709; T426 and A712; T426 and T714; T426 and A790; T426 andA841; T426 and T862; A433 and A435; A433 and T530; A433 and C572; A433and T596; A433 and T617; A433 and T688; A433 and A696; A433 and T702;A433 and A709; A433 and A712; A433 and T714; A433 and A790; A433 andA841; A433 and T862; A435 and T530; A435 and C572; A435 and T596; A435and T617; A435 and T688; A435 and A696; A435 and T702; A435 and A709;A435 and A712; A435 and T714; A435 and A790; A435 and A841; A435 andT862; T530 and C572; T530 and T596; T530 and T617; T530 and T688; T530and A696; T530 and T702; T530 and A709; T530 and A712; T530 and T714;T530 and A790; T530 and A841; T530 and T862; C572 and T596; C572 andT617; C572 and T688; C572 and A696; C572 and T702; C572 and A709; C572and A712; C572 and T714; C572 and A790; C572 and A841; C572 and T862;T596 and T617; T596 and T688; T596 and A696; T596 and T702; T596 andA709; T596 and A712; T596 and T714; T596 and A790; T596 and A841; T596and T862; T617 and T688; T617 and A696; T617 and T702; T617 and A709;T617 and A712; T617 and T714; T617 and A790; T617 and A841; T617 andT862; T688 and A696; T688 and T702; T688 and A709; T688 and A712; T688and T714; T688 and A790; T688 and A841; T688 and T862; A696 and T702;A696 and A709; A696 and A712; A696 and T714; A696 and A790; A696 andA841; A696 and T862; T702 and A709; T702 and A712; T702 and T714; T702and A790; T702 and A841; T702 and T862; A709 and A712; A709 and T714;A709 and A790; A709 and A841; A709 and T862; A712 and T714; A712 andA790; A712 and A841; A712 and T862; T714 and A790; T714 and A841; T714and T862; A790 and A841; A790 and T862; or A841 and T862.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations at positions corresponding to thefollowing positions relative to SEQ ID NO: 28 can be present in apromoter sequence: T146, C154, and T303; T146, C154, and T426; T146,C154, and A433; T146, C154, and A435; T146, C154, and T530; T146, C154,and C572; T146, C154, and T596; T146, C154, and T617; T146, C154, andT688; T146, C154, and A696; T146, C154, and T702; T146, C154, and A709;T146, C154, and A712; T146, C154, and T714; T146, C154, and A790; T146,C154, and A841; T146, C154, and T862; T146, T303, and T426; T146, T303,and A433; T146, T303, and A435; T146, T303, and T530; T146, T303, andC572; T146, T303, and T596; T146, T303, and T617; T146, T303, and T688;T146, T303, and A696; T146, T303, and T702; T146, T303, and A709; T146,T303, and A712; T146, T303, and T714; T146, T303, and A790; T146, T303,and A841; T146, T303, and T862; T146, T426, and A433; T146, T426, andA435; T146, T426, and T530; T146, T426, and C572; T146, T426, and T596;T146, T426, and T617; T146, T426, and T688; T146, T426, and A696; T146,T426, and T702; T146, T426, and A709; T146, T426, and A712; T146, T426,and T714; T146, T426, and A790; T146, T426, and A841; T146, T426, andT862; T146, A433, and A435; T146, A433, and T530; T146, A433, and C572;T146, A433, and T596; T146, A433, and T617; T146, A433, and T688; T146,A433, and A696; T146, A433, and T702; T146, A433, and A709; T146, A433,and A712; T146, A433, and T714; T146, A433, and A790; T146, A433, andA841; T146, A433, and T862; T146, A435, and T530; T146, A435, and C572;T146, A435, and T596; T146, A435, and T617; T146, A435, and T688; T146,A435, and A696; T146, A435, and T702; T146, A435, and A709; T146, A435,and A712; T146, A435, and T714; T146, A435, and A790; T146, A435, andA841; T146, A435, and T862; T146, T530, and C572; T146, T530, and T596;T146, T530, and T617; T146, T530, and T688; T146, T530, and A696; T146,T530, and T702; T146, T530, and A709; T146, T530, and A712; T146, T530,and T714; T146, T530, and A790; T146, T530, and A841; T146, T530, andT862; T146, C572, and T596; T146, C572, and T617; T146, C572, and T688;T146, C572, and A696; T146, C572, and T702; T146, C572, and A709; T146,C572, and A712; T146, C572, and T714; T146, C572, and A790; T146, C572,and A841; T146, C572, and T862; T146, T596, and T617; T146, T596, andT688; T146, T596, and A696; T146, T596, and T702; T146, T596, and A709;T146, T596, and A712; T146, T596, and T714; T146, T596, and A790; T146,T596, and A841; T146, T596, and T862; T146, T617, and T688; T146, T617,and A696; T146, T617, and T702; T146, T617, and A709; T146, T617, andA712; T146, T617, and T714; T146, T617, and A790; T146, T617, and A841;T146, T617, and T862; T146, T688, and A696; T146, T688, and T702; T146,T688, and A709; T146, T688, and A712; T146, T688, and T714; T146, T688,and A790; T146, T688, and A841; T146, T688, and T862; T146, A696, andT702; T146, A696, and A709; T146, A696, and A712; T146, A696, and T714;T146, A696, and A790; T146, A696, and A841; T146, A696, and T862; T146,T702, and A709; T146, T702, and A712; T146, T702, and T714; T146, T702,and A790; T146, T702, and A841; T146, T702, and T862; T146, A709, andA712; T146, A709, and T714; T146, A709, and A790; T146, A709, and A841;T146, A709, and T862; T146, A712, and T714; T146, A712, and A790; T146,A712, and A841; T146, A712, and T862; T146, T714, and A790; T146, T714,and A841; T146, T714, and T862; T146, A790, and A841; T146, A790, andT862; T146, A841, and T862; C154, T303, and T426; C154, T303, and A433;C154, T303, and A435; C154, T303, and T530; C154, T303, and C572; C154,T303, and T596; C154, T303, and T617; C154, T303, and T688; C154, T303,and A696; C154, T303, and T702; C154, T303, and A709; C154, T303, andA712; C154, T303, and T714; C154, T303, and A790; C154, T303, and A841;C154, T303, and T862; C154, T426, and A433; C154, T426, and A435; C154,T426, and T530; C154, T426, and C572; C154, T426, and T596; C154, T426,and T617; C154, T426, and T688; C154, T426, and A696; C154, T426, andT702; C154, T426, and A709; C154, T426, and A712; C154, T426, and T714;C154, T426, and A790; C154, T426, and A841; C154, T426, and T862; C154,A433, and A435; C154, A433, and T530; C154, A433, and C572; C154, A433,and T596; C154, A433, and T617; C154, A433, and T688; C154, A433, andA696; C154, A433, and T702; C154, A433, and A709; C154, A433, and A712;C154, A433, and T714; C154, A433, and A790; C154, A433, and A841; C154,A433, and T862; C154, A435, and T530; C154, A435, and C572; C154, A435,and T596; C154, A435, and T617; C154, A435, and T688; C154, A435, andA696; C154, A435, and T702; C154, A435, and A709; C154, A435, and A712;C154, A435, and T714; C154, A435, and A790; C154, A435, and A841; C154,A435, and T862; C154, T530, and C572; C154, T530, and T596; C154, T530,and T617; C154, T530, and T688; C154, T530, and A696; C154, T530, andT702; C154, T530, and A709; C154, T530, and A712; C154, T530, and T714;C154, T530, and A790; C154, T530, and A841; C154, T530, and T862; C154,C572, and T596; C154, C572, and T617; C154, C572, and T688; C154, C572,and A696; C154, C572, and T702; C154, C572, and A709; C154, C572, andA712; C154, C572, and T714; C154, C572, and A790; C154, C572, and A841;C154, C572, and T862; C154, T596, and T617; C154, T596, and T688; C154,T596, and A696; C154, T596, and T702; C154, T596, and A709; C154, T596,and A712; C154, T596, and T714; C154, T596, and A790; C154, T596, andA841; C154, T596, and T862; C154, T617, and T688; C154, T617, and A696;C154, T617, and T702; C154, T617, and A709; C154, T617, and A712; C154,T617, and T714; C154, T617, and A790; C154, T617, and A841; C154, T617,and T862; C154, T688, and A696; C154, T688, and T702; C154, T688, andA709; C154, T688, and A712; C154, T688, and T714; C154, T688, and A790;C154, T688, and A841; C154, T688, and T862; C154, A696, and T702; C154,A696, and A709; C154, A696, and A712; C154, A696, and T714; C154, A696,and A790; C154, A696, and A841; C154, A696, and T862; C154, T702, andA709; C154, T702, and A712; C154, T702, and T714; C154, T702, and A790;C154, T702, and A841; C154, T702, and T862; C154, A709, and A712; C154,A709, and T714; C154, A709, and A790; C154, A709, and A841; C154, A709,and T862; C154, A712, and T714; C154, A712, and A790; C154, A712, andA841; C154, A712, and T862; C154, T714, and A790; C154, T714, and A841;C154, T714, and T862; C154, A790, and A841; C154, A790, and T862; C154,A841, and T862; T303, T426, and A433; T303, T426, and A435; T303, T426,and T530; T303, T426, and C572; T303, T426, and T596; T303, T426, andT617; T303, T426, and T688; T303, T426, and A696; T303, T426, and T702;T303, T426, and A709; T303, T426, and A712; T303, T426, and T714; T303,T426, and A790; T303, T426, and A841; T303, T426, and T862; T303, A433,and A435; T303, A433, and T530; T303, A433, and C572; T303, A433, andT596; T303, A433, and T617; T303, A433, and T688; T303, A433, and A696;T303, A433, and T702; T303, A433, and A709; T303, A433, and A712; T303,A433, and T714; T303, A433, and A790; T303, A433, and A841; T303, A433,and T862; T303, A435, and T530; T303, A435, and C572; T303, A435, andT596; T303, A435, and T617; T303, A435, and T688; T303, A435, and A696;T303, A435, and T702; T303, A435, and A709; T303, A435, and A712; T303,A435, and T714; T303, A435, and A790; T303, A435, and A841; T303, A435,and T862; T303, T530, and C572; T303, T530, and T596; T303, T530, andT617; T303, T530, and T688; T303, T530, and A696; T303, T530, and T702;T303, T530, and A709; T303, T530, and A712; T303, T530, and T714; T303,T530, and A790; T303, T530, and A841; T303, T530, and T862; T303, C572,and T596; T303, C572, and T617; T303, C572, and T688; T303, C572, andA696; T303, C572, and T702; T303, C572, and A709; T303, C572, and A712;T303, C572, and T714; T303, C572, and A790; T303, C572, and A841; T303,C572, and T862; T303, T596, and T617; T303, T596, and T688; T303, T596,and A696; T303, T596, and T702; T303, T596, and A709; T303, T596, andA712; T303, T596, and T714; T303, T596, and A790; T303, T596, and A841;T303, T596, and T862; T303, T617, and T688; T303, T617, and A696; T303,T617, and T702; T303, T617, and A709; T303, T617, and A712; T303, T617,and T714; T303, T617, and A790; T303, T617, and A841; T303, T617, andT862; T303, T688, and A696; T303, T688, and T702; T303, T688, and A709;T303, T688, and A712; T303, T688, and T714; T303, T688, and A790; T303,T688, and A841; T303, T688, and T862; T303, A696, and T702; T303, A696,and A709; T303, A696, and A712; T303, A696, and T714; T303, A696, andA790; T303, A696, and A841; T303, A696, and T862; T303, T702, and A709;T303, T702, and A712; T303, T702, and T714; T303, T702, and A790; T303,T702, and A841; T303, T702, and T862; T303, A709, and A712; T303, A709,and T714; T303, A709, and A790; T303, A709, and A841; T303, A709, andT862; T303, A712, and T714; T303, A712, and A790; T303, A712, and A841;T303, A712, and T862; T303, T714, and A790; T303, T714, and A841; T303,T714, and T862; T303, A790, and A841; T303, A790, and T862; T303, A841,and T862; T426, A433, and A435; T426, A433, and T530; T426, A433, andC572; T426, A433, and T596; T426, A433, and T617; T426, A433, and T688;T426, A433, and A696; T426, A433, and T702; T426, A433, and A709; T426,A433, and A712; T426, A433, and T714; T426, A433, and A790; T426, A433,and A841; T426, A433, and T862; T426, A435, and T530; T426, A435, andC572; T426, A435, and T596; T426, A435, and T617; T426, A435, and T688;T426, A435, and A696; T426, A435, and T702; T426, A435, and A709; T426,A435, and A712; T426, A435, and T714; T426, A435, and A790; T426, A435,and A841; T426, A435, and T862; T426, T530, and C572; T426, T530, andT596; T426, T530, and T617; T426, T530, and T688; T426, T530, and A696;T426, T530, and T702; T426, T530, and A709; T426, T530, and A712; T426,T530, and T714; T426, T530, and A790; T426, T530, and A841; T426, T530,and T862; T426, C572, and T596; T426, C572, and T617; T426, C572, andT688; T426, C572, and A696; T426, C572, and T702; T426, C572, and A709;T426, C572, and A712; T426, C572, and T714; T426, C572, and A790; T426,C572, and A841; T426, C572, and T862; T426, T596, and T617; T426, T596,and T688; T426, T596, and A696; T426, T596, and T702; T426, T596, andA709; T426, T596, and A712; T426, T596, and T714; T426, T596, and A790;T426, T596, and A841; T426, T596, and T862; T426, T617, and T688; T426,T617, and A696; T426, T617, and T702; T426, T617, and A709; T426, T617,and A712; T426, T617, and T714; T426, T617, and A790; T426, T617, andA841; T426, T617, and T862; T426, T688, and A696; T426, T688, and T702;T426, T688, and A709; T426, T688, and A712; T426, T688, and T714; T426,T688, and A790; T426, T688, and A841; T426, T688, and T862; T426, A696,and T702; T426, A696, and A709; T426, A696, and A712; T426, A696, andT714; T426, A696, and A790; T426, A696, and A841; T426, A696, and T862;T426, T702, and A709; T426, T702, and A712; T426, T702, and T714; T426,T702, and A790; T426, T702, and A841; T426, T702, and T862; T426, A709,and A712; T426, A709, and T714; T426, A709, and A790; T426, A709, andA841; T426, A709, and T862; T426, A712, and T714; T426, A712, and A790;T426, A712, and A841; T426, A712, and T862; T426, T714, and A790; T426,T714, and A841; T426, T714, and T862; T426, A790, and A841; T426, A790,and T862; T426, A841, and T862; A433, A435, and T530; A433, A435, andC572; A433, A435, and T596; A433, A435, and T617; A433, A435, and T688;A433, A435, and A696; A433, A435, and T702; A433, A435, and A709; A433,A435, and A712; A433, A435, and T714; A433, A435, and A790; A433, A435,and A841; A433, A435, and T862; A433, T530, and C572; A433, T530, andT596; A433, T530, and T617; A433, T530, and T688; A433, T530, and A696;A433, T530, and T702; A433, T530, and A709; A433, T530, and A712; A433,T530, and T714; A433, T530, and A790; A433, T530, and A841; A433, T530,and T862; A433, C572, and T596; A433, C572, and T617; A433, C572, andT688; A433, C572, and A696; A433, C572, and T702; A433, C572, and A709;A433, C572, and A712; A433, C572, and T714; A433, C572, and A790; A433,C572, and A841; A433, C572, and T862; A433, T596, and T617; A433, T596,and T688; A433, T596, and A696; A433, T596, and T702; A433, T596, andA709; A433, T596, and A712; A433, T596, and T714; A433, T596, and A790;A433, T596, and A841; A433, T596, and T862; A433, T617, and T688; A433,T617, and A696; A433, T617, and T702; A433, T617, and A709; A433, T617,and A712; A433, T617, and T714; A433, T617, and A790; A433, T617, andA841; A433, T617, and T862; A433, T688, and A696; A433, T688, and T702;A433, T688, and A709; A433, T688, and A712; A433, T688, and T714; A433,T688, and A790; A433, T688, and A841; A433, T688, and T862; A433, A696,and T702; A433, A696, and A709; A433, A696, and A712; A433, A696, andT714; A433, A696, and A790; A433, A696, and A841; A433, A696, and T862;A433, T702, and A709; A433, T702, and A712; A433, T702, and T714; A433,T702, and A790; A433, T702, and A841; A433, T702, and T862; A433, A709,and A712; A433, A709, and T714; A433, A709, and A790; A433, A709, andA841; A433, A709, and T862; A433, A712, and T714; A433, A712, and A790;A433, A712, and A841; A433, A712, and T862; A433, T714, and A790; A433,T714, and A841; A433, T714, and T862; A433, A790, and A841; A433, A790,and T862; A433, A841, and T862; A435, T530, and C572; A435, T530, andT596; A435, T530, and T617; A435, T530, and T688; A435, T530, and A696;A435, T530, and T702; A435, T530, and A709; A435, T530, and A712; A435,T530, and T714; A435, T530, and A790; A435, T530, and A841; A435, T530,and T862; A435, C572, and T596; A435, C572, and T617; A435, C572, andT688; A435, C572, and A696; A435, C572, and T702; A435, C572, and A709;A435, C572, and A712; A435, C572, and T714; A435, C572, and A790; A435,C572, and A841; A435, C572, and T862; A435, T596, and T617; A435, T596,and T688; A435, T596, and A696; A435, T596, and T702; A435, T596, andA709; A435, T596, and A712; A435, T596, and T714; A435, T596, and A790;A435, T596, and A841; A435, T596, and T862; A435, T617, and T688; A435,T617, and A696; A435, T617, and T702; A435, T617, and A709; A435, T617,and A712; A435, T617, and T714; A435, T617, and A790; A435, T617, andA841; A435, T617, and T862; A435, T688, and A696; A435, T688, and T702;A435, T688, and A709; A435, T688, and A712; A435, T688, and T714; A435,T688, and A790; A435, T688, and A841; A435, T688, and T862; A435, A696,and T702; A435, A696, and A709; A435, A696, and A712; A435, A696, andT714; A435, A696, and A790; A435, A696, and A841; A435, A696, and T862;A435, T702, and A709; A435, T702, and A712; A435, T702, and T714; A435,T702, and A790; A435, T702, and A841; A435, T702, and T862; A435, A709,and A712; A435, A709, and T714; A435, A709, and A790; A435, A709, andA841; A435, A709, and T862; A435, A712, and T714; A435, A712, and A790;A435, A712, and A841; A435, A712, and T862; A435, T714, and A790; A435,T714, and A841; A435, T714, and T862; A435, A790, and A841; A435, A790,and T862; A435, A841, and T862; T530, C572, and T596; T530, C572, andT617; T530, C572, and T688; T530, C572, and A696; T530, C572, and T702;T530, C572, and A709; T530, C572, and A712; T530, C572, and T714; T530,C572, and A790; T530, C572, and A841; T530, C572, and T862; T530, T596,and T617; T530, T596, and T688; T530, T596, and A696; T530, T596, andT702; T530, T596, and A709; T530, T596, and A712; T530, T596, and T714;T530, T596, and A790; T530, T596, and A841; T530, T596, and T862; T530,T617, and T688; T530, T617, and A696; T530, T617, and T702; T530, T617,and A709; T530, T617, and A712; T530, T617, and T714; T530, T617, andA790; T530, T617, and A841; T530, T617, and T862; T530, T688, and A696;T530, T688, and T702; T530, T688, and A709; T530, T688, and A712; T530,T688, and T714; T530, T688, and A790; T530, T688, and A841; T530, T688,and T862; T530, A696, and T702; T530, A696, and A709; T530, A696, andA712; T530, A696, and T714; T530, A696, and A790; T530, A696, and A841;T530, A696, and T862; T530, T702, and A709; T530, T702, and A712; T530,T702, and T714; T530, T702, and A790; T530, T702, and A841; T530, T702,and T862; T530, A709, and A712; T530, A709, and T714; T530, A709, andA790; T530, A709, and A841; T530, A709, and T862; T530, A712, and T714;T530, A712, and A790; T530, A712, and A841; T530, A712, and T862; T530,T714, and A790; T530, T714, and A841; T530, T714, and T862; T530, A790,and A841; T530, A790, and T862; T530, A841, and T862; C572, T596, andT617; C572, T596, and T688; C572, T596, and A696; C572, T596, and T702;C572, T596, and A709; C572, T596, and A712; C572, T596, and T714; C572,T596, and A790; C572, T596, and A841; C572, T596, and T862; C572, T617,and T688; C572, T617, and A696; C572, T617, and T702; C572, T617, andA709; C572, T617, and A712; C572, T617, and T714; C572, T617, and A790;C572, T617, and A841; C572, T617, and T862; C572, T688, and A696; C572,T688, and T702; C572, T688, and A709; C572, T688, and A712; C572, T688,and T714; C572, T688, and A790; C572, T688, and A841; C572, T688, andT862; C572, A696, and T702; C572, A696, and A709; C572, A696, and A712;C572, A696, and T714; C572, A696, and A790; C572, A696, and A841; C572,A696, and T862; C572, T702, and A709; C572, T702, and A712; C572, T702,and T714; C572, T702, and A790; C572, T702, and A841; C572, T702, andT862; C572, A709, and A712; C572, A709, and T714; C572, A709, and A790;C572, A709, and A841; C572, A709, and T862; C572, A712, and T714; C572,A712, and A790; C572, A712, and A841; C572, A712, and T862; C572, T714,and A790; C572, T714, and A841; C572, T714, and T862; C572, A790, andA841; C572, A790, and T862; C572, A841, and T862; T596, T617, and T688;T596, T617, and A696; T596, T617, and T702; T596, T617, and A709; T596,T617, and A712; T596, T617, and T714; T596, T617, and A790; T596, T617,and A841; T596, T617, and T862; T596, T688, and A696; T596, T688, andT702; T596, T688, and A709; T596, T688, and A712; T596, T688, and T714;T596, T688, and A790; T596, T688, and A841; T596, T688, and T862; T596,A696, and T702; T596, A696, and A709; T596, A696, and A712; T596, A696,and T714; T596, A696, and A790; T596, A696, and A841; T596, A696, andT862; T596, T702, and A709; T596, T702, and A712; T596, T702, and T714;T596, T702, and A790; T596, T702, and A841; T596, T702, and T862; T596,A709, and A712; T596, A709, and T714; T596, A709, and A790; T596, A709,and A841; T596, A709, and T862; T596, A712, and T714; T596, A712, andA790; T596, A712, and A841; T596, A712, and T862; T596, T714, and A790;T596, T714, and A841; T596, T714, and T862; T596, A790, and A841; T596,A790, and T862; T596, A841, and T862; T617, T688, and A696; T617, T688,and T702; T617, T688, and A709; T617, T688, and A712; T617, T688, andT714; T617, T688, and A790; T617, T688, and A841; T617, T688, and T862;T617, A696, and T702; T617, A696, and A709; T617, A696, and A712; T617,A696, and T714; T617, A696, and A790; T617, A696, and A841; T617, A696,and T862; T617, T702, and A709; T617, T702, and A712; T617, T702, andT714; T617, T702, and A790; T617, T702, and A841; T617, T702, and T862;T617, A709, and A712; T617, A709, and T714; T617, A709, and A790; T617,A709, and A841; T617, A709, and T862; T617, A712, and T714; T617, A712,and A790; T617, A712, and A841; T617, A712, and T862; T617, T714, andA790; T617, T714, and A841; T617, T714, and T862; T617, A790, and A841;T617, A790, and T862; T617, A841, and T862; T688, A696, and T702; T688,A696, and A709; T688, A696, and A712; T688, A696, and T714; T688, A696,and A790; T688, A696, and A841; T688, A696, and T862; T688, T702, andA709; T688, T702, and A712; T688, T702, and T714; T688, T702, and A790;T688, T702, and A841; T688, T702, and T862; T688, A709, and A712; T688,A709, and T714; T688, A709, and A790; T688, A709, and A841; T688, A709,and T862; T688, A712, and T714; T688, A712, and A790; T688, A712, andA841; T688, A712, and T862; T688, T714, and A790; T688, T714, and A841;T688, T714, and T862; T688, A790, and A841; T688, A790, and T862; T688,A841, and T862; A696, T702, and A709; A696, T702, and A712; A696, T702,and T714; A696, T702, and A790; A696, T702, and A841; A696, T702, andT862; A696, A709, and A712; A696, A709, and T714; A696, A709, and A790;A696, A709, and A841; A696, A709, and T862; A696, A712, and T714; A696,A712, and A790; A696, A712, and A841; A696, A712, and T862; A696, T714,and A790; A696, T714, and A841; A696, T714, and T862; A696, A790, andA841; A696, A790, and T862; A696, A841, and T862; T702, A709, and A712;T702, A709, and T714; T702, A709, and A790; T702, A709, and A841; T702,A709, and T862; T702, A712, and T714; T702, A712, and A790; T702, A712,and A841; T702, A712, and T862; T702, T714, and A790; T702, T714, andA841; T702, T714, and T862; T702, A790, and A841; T702, A790, and T862;T702, A841, and T862; A709, A712, and T714; A709, A712, and A790; A709,A712, and A841; A709, A712, and T862; A709, T714, and A790; A709, T714,and A841; A709, T714, and T862; A709, A790, and A841; A709, A790, andT862; A709, A841, and T862; A712, T714, and A790; A712, T714, and A841;A712, T714, and T862; A712, A790, and A841; A712, A790, and T862; A712,A841, and T862; T714, A790, and A841; T714, A790, and T862; T714, A841,and T862; or A790, A841, and T862.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: T688 and A696; T688 and T702; T688 and A712; T688 and T714;A696 and T702; A696 and A712; A696 and T714; T702 and A712; T702 andT714; or A712 and T714.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations at positions corresponding to thefollowing positions relative to SEQ ID NO: 28 can be present in apromoter sequence: T688, A696, and T702; T688, A696, and A712; T688,A696, and T714; T688, T702, and A712; T688, T702, and T714; T688, A712,and T714; A696, T702, and A712; A696, T702, and T714; A696, A712, andT714; or T702, A712, and T714.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include four mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, four mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: T688, A696, T702, and A712; T688, A696, T702, and T714; T688,A696, A712, and T714; T688, T702, A712, and T714; or A696, T702, A712,and T714.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include five mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, five mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: T688, A696, T702, A712, and T714.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: 146 and 154; 146 and 303; 146 and 426; 146 and 433; 146 and435; 146 and 530; 146 and 572; 146 and 596; 146 and 617; 146 and 688;146 and 696; 146 and 702; 146 and 709; 146 and A712; 146 and 714; 146and 790; 146 and 841; 146 and 862; 154 and 303; 154 and 426; 154 and433; 154 and 435; 154 and 530; 154 and 572; 154 and 596; 154 and 617;154 and 688; 154 and 696; 154 and 702; 154 and 709; 154 and A712; 154and 714; 154 and 790; 154 and 841; 154 and 862; 303 and 426; 303 and433; 303 and 435; 303 and 530; 303 and 572; 303 and 596; 303 and 617;303 and 688; 303 and 696; 303 and 702; 303 and 709; 303 and A712; 303and 714; 303 and 790; 303 and 841; 303 and 862; 426 and 433; 426 and435; 426 and 530; 426 and 572; 426 and 596; 426 and 617; 426 and 688;426 and 696; 426 and 702; 426 and 709; 426 and A712; 426 and 714; 426and 790; 426 and 841; 426 and 862; 433 and 435; 433 and 530; 433 and572; 433 and 596; 433 and 617; 433 and 688; 433 and 696; 433 and 702;433 and 709; 433 and A712; 433 and 714; 433 and 790; 433 and 841; 433and 862; 435 and 530; 435 and 572; 435 and 596; 435 and 617; 435 and688; 435 and 696; 435 and 702; 435 and 709; 435 and A712; 435 and 714;435 and 790; 435 and 841; 435 and 862; 530 and 572; 530 and 596; 530 and617; 530 and 688; 530 and 696; 530 and 702; 530 and 709; 530 and A712;530 and 714; 530 and 790; 530 and 841; 530 and 862; 572 and 596; 572 and617; 572 and 688; 572 and 696; 572 and 702; 572 and 709; 572 and A712;572 and 714; 572 and 790; 572 and 841; 572 and 862; 596 and 617; 596 and688; 596 and 696; 596 and 702; 596 and 709; 596 and A712; 596 and 714;596 and 790; 596 and 841; 596 and 862; 617 and 688; 617 and 696; 617 and702; 617 and 709; 617 and A712; 617 and 714; 617 and 790; 617 and 841;617 and 862; 688 and 696; 688 and 702; 688 and 709; 688 and A712; 688and 714; 688 and 790; 688 and 841; 688 and 862; 696 and 702; 696 and709; 696 and A712; 696 and 714; 696 and 790; 696 and 841; 696 and 862;702 and 709; 702 and A712; 702 and 714; 702 and 790; 702 and 841; 702and 862; 709 and A712; 709 and 714; 709 and 790; 709 and 841; 709 and862; A712 and 714; A712 and 790; A712 and 841; A712 and 862; 714 and790; 714 and 841; 714 and 862; 790 and 841; 790 and 862; or 841 and 862.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations at positions corresponding to thefollowing positions relative to SEQ ID NO: 28 can be present in apromoter sequence: 146, 154, and 303; 146, 154, and 426; 146, 154, and433; 146, 154, and 435; 146, 154, and 530; 146, 154, and 572; 146, 154,and 596; 146, 154, and 617; 146, 154, and 688; 146, 154, and 696; 146,154, and 702; 146, 154, and 709; 146, 154, and A712; 146, 154, and 714;146, 154, and 790; 146, 154, and 841; 146, 154, and 862; 146, 303, and426; 146, 303, and 433; 146, 303, and 435; 146, 303, and 530; 146, 303,and 572; 146, 303, and 596; 146, 303, and 617; 146, 303, and 688; 146,303, and 696; 146, 303, and 702; 146, 303, and 709; 146, 303, and A712;146, 303, and 714; 146, 303, and 790; 146, 303, and 841; 146, 303, and862; 146, 426, and 433; 146, 426, and 435; 146, 426, and 530; 146, 426,and 572; 146, 426, and 596; 146, 426, and 617; 146, 426, and 688; 146,426, and 696; 146, 426, and 702; 146, 426, and 709; 146, 426, and A712;146, 426, and 714; 146, 426, and 790; 146, 426, and 841; 146, 426, and862; 146, 433, and 435; 146, 433, and 530; 146, 433, and 572; 146, 433,and 596; 146, 433, and 617; 146, 433, and 688; 146, 433, and 696; 146,433, and 702; 146, 433, and 709; 146, 433, and A712; 146, 433, and 714;146, 433, and 790; 146, 433, and 841; 146, 433, and 862; 146, 435, and530; 146, 435, and 572; 146, 435, and 596; 146, 435, and 617; 146, 435,and 688; 146, 435, and 696; 146, 435, and 702; 146, 435, and 709; 146,435, and A712; 146, 435, and 714; 146, 435, and 790; 146, 435, and 841;146, 435, and 862; 146, 530, and 572; 146, 530, and 596; 146, 530, and617; 146, 530, and 688; 146, 530, and 696; 146, 530, and 702; 146, 530,and 709; 146, 530, and A712; 146, 530, and 714; 146, 530, and 790; 146,530, and 841; 146, 530, and 862; 146, 572, and 596; 146, 572, and 617;146, 572, and 688; 146, 572, and 696; 146, 572, and 702; 146, 572, and709; 146, 572, and A712; 146, 572, and 714; 146, 572, and 790; 146, 572,and 841; 146, 572, and 862; 146, 596, and 617; 146, 596, and 688; 146,596, and 696; 146, 596, and 702; 146, 596, and 709; 146, 596, and A712;146, 596, and 714; 146, 596, and 790; 146, 596, and 841; 146, 596, and862; 146, 617, and 688; 146, 617, and 696; 146, 617, and 702; 146, 617,and 709; 146, 617, and A712; 146, 617, and 714; 146, 617, and 790; 146,617, and 841; 146, 617, and 862; 146, 688, and 696; 146, 688, and 702;146, 688, and 709; 146, 688, and A712; 146, 688, and 714; 146, 688, and790; 146, 688, and 841; 146, 688, and 862; 146, 696, and 702; 146, 696,and 709; 146, 696, and A712; 146, 696, and 714; 146, 696, and 790; 146,696, and 841; 146, 696, and 862; 146, 702, and 709; 146, 702, and A712;146, 702, and 714; 146, 702, and 790; 146, 702, and 841; 146, 702, and862; 146, 709, and A712; 146, 709, and 714; 146, 709, and 790; 146, 709,and 841; 146, 709, and 862; 146, A712, and 714; 146, A712, and 790; 146,A712, and 841; 146, A712, and 862; 146, 714, and 790; 146, 714, and 841;146, 714, and 862; 146, 790, and 841; 146, 790, and 862; 146, 841, and862; 154, 303, and 426; 154, 303, and 433; 154, 303, and 435; 154, 303,and 530; 154, 303, and 572; 154, 303, and 596; 154, 303, and 617; 154,303, and 688; 154, 303, and 696; 154, 303, and 702; 154, 303, and 709;154, 303, and A712; 154, 303, and 714; 154, 303, and 790; 154, 303, and841; 154, 303, and 862; 154, 426, and 433; 154, 426, and 435; 154, 426,and 530; 154, 426, and 572; 154, 426, and 596; 154, 426, and 617; 154,426, and 688; 154, 426, and 696; 154, 426, and 702; 154, 426, and 709;154, 426, and A712; 154, 426, and 714; 154, 426, and 790; 154, 426, and841; 154, 426, and 862; 154, 433, and 435; 154, 433, and 530; 154, 433,and 572; 154, 433, and 596; 154, 433, and 617; 154, 433, and 688; 154,433, and 696; 154, 433, and 702; 154, 433, and 709; 154, 433, and A712;154, 433, and 714; 154, 433, and 790; 154, 433, and 841; 154, 433, and862; 154, 435, and 530; 154, 435, and 572; 154, 435, and 596; 154, 435,and 617; 154, 435, and 688; 154, 435, and 696; 154, 435, and 702; 154,435, and 709; 154, 435, and A712; 154, 435, and 714; 154, 435, and 790;154, 435, and 841; 154, 435, and 862; 154, 530, and 572; 154, 530, and596; 154, 530, and 617; 154, 530, and 688; 154, 530, and 696; 154, 530,and 702; 154, 530, and 709; 154, 530, and A712; 154, 530, and 714; 154,530, and 790; 154, 530, and 841; 154, 530, and 862; 154, 572, and 596;154, 572, and 617; 154, 572, and 688; 154, 572, and 696; 154, 572, and702; 154, 572, and 709; 154, 572, and A712; 154, 572, and 714; 154, 572,and 790; 154, 572, and 841; 154, 572, and 862; 154, 596, and 617; 154,596, and 688; 154, 596, and 696; 154, 596, and 702; 154, 596, and 709;154, 596, and A712; 154, 596, and 714; 154, 596, and 790; 154, 596, and841; 154, 596, and 862; 154, 617, and 688; 154, 617, and 696; 154, 617,and 702; 154, 617, and 709; 154, 617, and A712; 154, 617, and 714; 154,617, and 790; 154, 617, and 841; 154, 617, and 862; 154, 688, and 696;154, 688, and 702; 154, 688, and 709; 154, 688, and A712; 154, 688, and714; 154, 688, and 790; 154, 688, and 841; 154, 688, and 862; 154, 696,and 702; 154, 696, and 709; 154, 696, and A712; 154, 696, and 714; 154,696, and 790; 154, 696, and 841; 154, 696, and 862; 154, 702, and 709;154, 702, and A712; 154, 702, and 714; 154, 702, and 790; 154, 702, and841; 154, 702, and 862; 154, 709, and A712; 154, 709, and 714; 154, 709,and 790; 154, 709, and 841; 154, 709, and 862; 154, A712, and 714; 154,A712, and 790; 154, A712, and 841; 154, A712, and 862; 154, 714, and790; 154, 714, and 841; 154, 714, and 862; 154, 790, and 841; 154, 790,and 862; 154, 841, and 862; 303, 426, and 433; 303, 426, and 435; 303,426, and 530; 303, 426, and 572; 303, 426, and 596; 303, 426, and 617;303, 426, and 688; 303, 426, and 696; 303, 426, and 702; 303, 426, and709; 303, 426, and A712; 303, 426, and 714; 303, 426, and 790; 303, 426,and 841; 303, 426, and 862; 303, 433, and 435; 303, 433, and 530; 303,433, and 572; 303, 433, and 596; 303, 433, and 617; 303, 433, and 688;303, 433, and 696; 303, 433, and 702; 303, 433, and 709; 303, 433, andA712; 303, 433, and 714; 303, 433, and 790; 303, 433, and 841; 303, 433,and 862; 303, 435, and 530; 303, 435, and 572; 303, 435, and 596; 303,435, and 617; 303, 435, and 688; 303, 435, and 696; 303, 435, and 702;303, 435, and 709; 303, 435, and A712; 303, 435, and 714; 303, 435, and790; 303, 435, and 841; 303, 435, and 862; 303, 530, and 572; 303, 530,and 596; 303, 530, and 617; 303, 530, and 688; 303, 530, and 696; 303,530, and 702; 303, 530, and 709; 303, 530, and A712; 303, 530, and 714;303, 530, and 790; 303, 530, and 841; 303, 530, and 862; 303, 572, and596; 303, 572, and 617; 303, 572, and 688; 303, 572, and 696; 303, 572,and 702; 303, 572, and 709; 303, 572, and A712; 303, 572, and 714; 303,572, and 790; 303, 572, and 841; 303, 572, and 862; 303, 596, and 617;303, 596, and 688; 303, 596, and 696; 303, 596, and 702; 303, 596, and709; 303, 596, and A712; 303, 596, and 714; 303, 596, and 790; 303, 596,and 841; 303, 596, and 862; 303, 617, and 688; 303, 617, and 696; 303,617, and 702; 303, 617, and 709; 303, 617, and A712; 303, 617, and 714;303, 617, and 790; 303, 617, and 841; 303, 617, and 862; 303, 688, and696; 303, 688, and 702; 303, 688, and 709; 303, 688, and A712; 303, 688,and 714; 303, 688, and 790; 303, 688, and 841; 303, 688, and 862; 303,696, and 702; 303, 696, and 709; 303, 696, and A712; 303, 696, and 714;303, 696, and 790; 303, 696, and 841; 303, 696, and 862; 303, 702, and709; 303, 702, and A712; 303, 702, and 714; 303, 702, and 790; 303, 702,and 841; 303, 702, and 862; 303, 709, and A712; 303, 709, and 714; 303,709, and 790; 303, 709, and 841; 303, 709, and 862; 303, A712, and 714;303, A712, and 790; 303, A712, and 841; 303, A712, and 862; 303, 714,and 790; 303, 714, and 841; 303, 714, and 862; 303, 790, and 841; 303,790, and 862; 303, 841, and 862; 426, 433, and 435; 426, 433, and 530;426, 433, and 572; 426, 433, and 596; 426, 433, and 617; 426, 433, and688; 426, 433, and 696; 426, 433, and 702; 426, 433, and 709; 426, 433,and A712; 426, 433, and 714; 426, 433, and 790; 426, 433, and 841; 426,433, and 862; 426, 435, and 530; 426, 435, and 572; 426, 435, and 596;426, 435, and 617; 426, 435, and 688; 426, 435, and 696; 426, 435, and702; 426, 435, and 709; 426, 435, and A712; 426, 435, and 714; 426, 435,and 790; 426, 435, and 841; 426, 435, and 862; 426, 530, and 572; 426,530, and 596; 426, 530, and 617; 426, 530, and 688; 426, 530, and 696;426, 530, and 702; 426, 530, and 709; 426, 530, and A712; 426, 530, and714; 426, 530, and 790; 426, 530, and 841; 426, 530, and 862; 426, 572,and 596; 426, 572, and 617; 426, 572, and 688; 426, 572, and 696; 426,572, and 702; 426, 572, and 709; 426, 572, and A712; 426, 572, and 714;426, 572, and 790; 426, 572, and 841; 426, 572, and 862; 426, 596, and617; 426, 596, and 688; 426, 596, and 696; 426, 596, and 702; 426, 596,and 709; 426, 596, and A712; 426, 596, and 714; 426, 596, and 790; 426,596, and 841; 426, 596, and 862; 426, 617, and 688; 426, 617, and 696;426, 617, and 702; 426, 617, and 709; 426, 617, and A712; 426, 617, and714; 426, 617, and 790; 426, 617, and 841; 426, 617, and 862; 426, 688,and 696; 426, 688, and 702; 426, 688, and 709; 426, 688, and A712; 426,688, and 714; 426, 688, and 790; 426, 688, and 841; 426, 688, and 862;426, 696, and 702; 426, 696, and 709; 426, 696, and A712; 426, 696, and714; 426, 696, and 790; 426, 696, and 841; 426, 696, and 862; 426, 702,and 709; 426, 702, and A712; 426, 702, and 714; 426, 702, and 790; 426,702, and 841; 426, 702, and 862; 426, 709, and A712; 426, 709, and 714;426, 709, and 790; 426, 709, and 841; 426, 709, and 862; 426, A712, and714; 426, A712, and 790; 426, A712, and 841; 426, A712, and 862; 426,714, and 790; 426, 714, and 841; 426, 714, and 862; 426, 790, and 841;426, 790, and 862; 426, 841, and 862; 433, 435, and 530; 433, 435, and572; 433, 435, and 596; 433, 435, and 617; 433, 435, and 688; 433, 435,and 696; 433, 435, and 702; 433, 435, and 709; 433, 435, and A712; 433,435, and 714; 433, 435, and 790; 433, 435, and 841; 433, 435, and 862;433, 530, and 572; 433, 530, and 596; 433, 530, and 617; 433, 530, and688; 433, 530, and 696; 433, 530, and 702; 433, 530, and 709; 433, 530,and A712; 433, 530, and 714; 433, 530, and 790; 433, 530, and 841; 433,530, and 862; 433, 572, and 596; 433, 572, and 617; 433, 572, and 688;433, 572, and 696; 433, 572, and 702; 433, 572, and 709; 433, 572, andA712; 433, 572, and 714; 433, 572, and 790; 433, 572, and 841; 433, 572,and 862; 433, 596, and 617; 433, 596, and 688; 433, 596, and 696; 433,596, and 702; 433, 596, and 709; 433, 596, and A712; 433, 596, and 714;433, 596, and 790; 433, 596, and 841; 433, 596, and 862; 433, 617, and688; 433, 617, and 696; 433, 617, and 702; 433, 617, and 709; 433, 617,and A712; 433, 617, and 714; 433, 617, and 790; 433, 617, and 841; 433,617, and 862; 433, 688, and 696; 433, 688, and 702; 433, 688, and 709;433, 688, and A712; 433, 688, and 714; 433, 688, and 790; 433, 688, and841; 433, 688, and 862; 433, 696, and 702; 433, 696, and 709; 433, 696,and A712; 433, 696, and 714; 433, 696, and 790; 433, 696, and 841; 433,696, and 862; 433, 702, and 709; 433, 702, and A712; 433, 702, and 714;433, 702, and 790; 433, 702, and 841; 433, 702, and 862; 433, 709, andA712; 433, 709, and 714; 433, 709, and 790; 433, 709, and 841; 433, 709,and 862; 433, A712, and 714; 433, A712, and 790; 433, A712, and 841;433, A712, and 862; 433, 714, and 790; 433, 714, and 841; 433, 714, and862; 433, 790, and 841; 433, 790, and 862; 433, 841, and 862; 435, 530,and 572; 435, 530, and 596; 435, 530, and 617; 435, 530, and 688; 435,530, and 696; 435, 530, and 702; 435, 530, and 709; 435, 530, and A712;435, 530, and 714; 435, 530, and 790; 435, 530, and 841; 435, 530, and862; 435, 572, and 596; 435, 572, and 617; 435, 572, and 688; 435, 572,and 696; 435, 572, and 702; 435, 572, and 709; 435, 572, and A712; 435,572, and 714; 435, 572, and 790; 435, 572, and 841; 435, 572, and 862;435, 596, and 617; 435, 596, and 688; 435, 596, and 696; 435, 596, and702; 435, 596, and 709; 435, 596, and A712; 435, 596, and 714; 435, 596,and 790; 435, 596, and 841; 435, 596, and 862; 435, 617, and 688; 435,617, and 696; 435, 617, and 702; 435, 617, and 709; 435, 617, and A712;435, 617, and 714; 435, 617, and 790; 435, 617, and 841; 435, 617, and862; 435, 688, and 696; 435, 688, and 702; 435, 688, and 709; 435, 688,and A712; 435, 688, and 714; 435, 688, and 790; 435, 688, and 841; 435,688, and 862; 435, 696, and 702; 435, 696, and 709; 435, 696, and A712;435, 696, and 714; 435, 696, and 790; 435, 696, and 841; 435, 696, and862; 435, 702, and 709; 435, 702, and A712; 435, 702, and 714; 435, 702,and 790; 435, 702, and 841; 435, 702, and 862; 435, 709, and A712; 435,709, and 714; 435, 709, and 790; 435, 709, and 841; 435, 709, and 862;435, A712, and 714; 435, A712, and 790; 435, A712, and 841; 435, A712,and 862; 435, 714, and 790; 435, 714, and 841; 435, 714, and 862; 435,790, and 841; 435, 790, and 862; 435, 841, and 862; 530, 572, and 596;530, 572, and 617; 530, 572, and 688; 530, 572, and 696; 530, 572, and702; 530, 572, and 709; 530, 572, and A712; 530, 572, and 714; 530, 572,and 790; 530, 572, and 841; 530, 572, and 862; 530, 596, and 617; 530,596, and 688; 530, 596, and 696; 530, 596, and 702; 530, 596, and 709;530, 596, and A712; 530, 596, and 714; 530, 596, and 790; 530, 596, and841; 530, 596, and 862; 530, 617, and 688; 530, 617, and 696; 530, 617,and 702; 530, 617, and 709; 530, 617, and A712; 530, 617, and 714; 530,617, and 790; 530, 617, and 841; 530, 617, and 862; 530, 688, and 696;530, 688, and 702; 530, 688, and 709; 530, 688, and A712; 530, 688, and714; 530, 688, and 790; 530, 688, and 841; 530, 688, and 862; 530, 696,and 702; 530, 696, and 709; 530, 696, and A712; 530, 696, and 714; 530,696, and 790; 530, 696, and 841; 530, 696, and 862; 530, 702, and 709;530, 702, and A712; 530, 702, and 714; 530, 702, and 790; 530, 702, and841; 530, 702, and 862; 530, 709, and A712; 530, 709, and 714; 530, 709,and 790; 530, 709, and 841; 530, 709, and 862; 530, A712, and 714; 530,A712, and 790; 530, A712, and 841; 530, A712, and 862; 530, 714, and790; 530, 714, and 841; 530, 714, and 862; 530, 790, and 841; 530, 790,and 862; 530, 841, and 862; 572, 596, and 617; 572, 596, and 688; 572,596, and 696; 572, 596, and 702; 572, 596, and 709; 572, 596, and A712;572, 596, and 714; 572, 596, and 790; 572, 596, and 841; 572, 596, and862; 572, 617, and 688; 572, 617, and 696; 572, 617, and 702; 572, 617,and 709; 572, 617, and A712; 572, 617, and 714; 572, 617, and 790; 572,617, and 841; 572, 617, and 862; 572, 688, and 696; 572, 688, and 702;572, 688, and 709; 572, 688, and A712; 572, 688, and 714; 572, 688, and790; 572, 688, and 841; 572, 688, and 862; 572, 696, and 702; 572, 696,and 709; 572, 696, and A712; 572, 696, and 714; 572, 696, and 790; 572,696, and 841; 572, 696, and 862; 572, 702, and 709; 572, 702, and A712;572, 702, and 714; 572, 702, and 790; 572, 702, and 841; 572, 702, and862; 572, 709, and A712; 572, 709, and 714; 572, 709, and 790; 572, 709,and 841; 572, 709, and 862; 572, A712, and 714; 572, A712, and 790; 572,A712, and 841; 572, A712, and 862; 572, 714, and 790; 572, 714, and 841;572, 714, and 862; 572, 790, and 841; 572, 790, and 862; 572, 841, and862; 596, 617, and 688; 596, 617, and 696; 596, 617, and 702; 596, 617,and 709; 596, 617, and A712; 596, 617, and 714; 596, 617, and 790; 596,617, and 841; 596, 617, and 862; 596, 688, and 696; 596, 688, and 702;596, 688, and 709; 596, 688, and A712; 596, 688, and 714; 596, 688, and790; 596, 688, and 841; 596, 688, and 862; 596, 696, and 702; 596, 696,and 709; 596, 696, and A712; 596, 696, and 714; 596, 696, and 790; 596,696, and 841; 596, 696, and 862; 596, 702, and 709; 596, 702, and A712;596, 702, and 714; 596, 702, and 790; 596, 702, and 841; 596, 702, and862; 596, 709, and A712; 596, 709, and 714; 596, 709, and 790; 596, 709,and 841; 596, 709, and 862; 596, A712, and 714; 596, A712, and 790; 596,A712, and 841; 596, A712, and 862; 596, 714, and 790; 596, 714, and 841;596, 714, and 862; 596, 790, and 841; 596, 790, and 862; 596, 841, and862; 617, 688, and 696; 617, 688, and 702; 617, 688, and 709; 617, 688,and A712; 617, 688, and 714; 617, 688, and 790; 617, 688, and 841; 617,688, and 862; 617, 696, and 702; 617, 696, and 709; 617, 696, and A712;617, 696, and 714; 617, 696, and 790; 617, 696, and 841; 617, 696, and862; 617, 702, and 709; 617, 702, and A712; 617, 702, and 714; 617, 702,and 790; 617, 702, and 841; 617, 702, and 862; 617, 709, and A712; 617,709, and 714; 617, 709, and 790; 617, 709, and 841; 617, 709, and 862;617, A712, and 714; 617, A712, and 790; 617, A712, and 841; 617, A712,and 862; 617, 714, and 790; 617, 714, and 841; 617, 714, and 862; 617,790, and 841; 617, 790, and 862; 617, 841, and 862; 688, 696, and 702;688, 696, and 709; 688, 696, and A712; 688, 696, and 714; 688, 696, and790; 688, 696, and 841; 688, 696, and 862; 688, 702, and 709; 688, 702,and A712; 688, 702, and 714; 688, 702, and 790; 688, 702, and 841; 688,702, and 862; 688, 709, and A712; 688, 709, and 714; 688, 709, and 790;688, 709, and 841; 688, 709, and 862; 688, A712, and 714; 688, A712, and790; 688, A712, and 841; 688, A712, and 862; 688, 714, and 790; 688,714, and 841; 688, 714, and 862; 688, 790, and 841; 688, 790, and 862;688, 841, and 862; 696, 702, and 709; 696, 702, and A712; 696, 702, and714; 696, 702, and 790; 696, 702, and 841; 696, 702, and 862; 696, 709,and A712; 696, 709, and 714; 696, 709, and 790; 696, 709, and 841; 696,709, and 862; 696, A712, and 714; 696, A712, and 790; 696, A712, and841; 696, A712, and 862; 696, 714, and 790; 696, 714, and 841; 696, 714,and 862; 696, 790, and 841; 696, 790, and 862; 696, 841, and 862; 702,709, and A712; 702, 709, and 714; 702, 709, and 790; 702, 709, and 841;702, 709, and 862; 702, A712, and 714; 702, A712, and 790; 702, A712,and 841; 702, A712, and 862; 702, 714, and 790; 702, 714, and 841; 702,714, and 862; 702, 790, and 841; 702, 790, and 862; 702, 841, and 862;709, A712, and 714; 709, A712, and 790; 709, A712, and 841; 709, A712,and 862; 709, 714, and 790; 709, 714, and 841; 709, 714, and 862; 709,790, and 841; 709, 790, and 862; 709, 841, and 862; A712, 714, and 790;A712, 714, and 841; A712, 714, and 862; A712, 790, and 841; A712, 790,and 862; A712, 841, and 862; 714, 790, and 841; 714, 790, and 862; 714,841, and 862; or 790, 841, and 862.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include two mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, two mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: 688 and 696; 688 and 702; 688 and A712; 688 and 714; 696 and702; 696 and A712; 696 and 714; 702 and A712; 702 and 714; or A712 and714.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include three mutations as compared to thenucleotide sequence of a reference promoter element. For example, insome embodiments, three mutations at positions corresponding to thefollowing positions relative to SEQ ID NO: 28 can be present in apromoter sequence: 688, 696, and 702; 688, 696, and A712; 688, 696, and714; 688, 702, and A712; 688, 702, and 714; 688, A712, and 714; 696,702, and A712; 696, 702, and 714; 696, A712, and 714; or 702, A712, and714.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include four mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, four mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: 688, 696, 702, and A712; 688, 696, 702, and 714; 688, 696,A712, and 714; 688, 702, A712, and 714; or 696, 702, A712, and 714.

In some embodiments, the nucleotide sequence of a promoter element asprovided herein can include five mutations as compared to the nucleotidesequence of a reference promoter element. For example, in someembodiments, five mutations at positions corresponding to the followingpositions relative to SEQ ID NO: 28 can be present in a promotersequence: 688, 696, 702, A712, and 714.

Nucleic acid molecules used in the methods described herein aretypically DNA, but RNA molecules can be used under the appropriatecircumstances. As used herein, “exogenous” refers to any nucleic acidsequence that is introduced into a cell from, for example, the same or adifferent organism or a nucleic acid generated synthetically (e.g., acodon-optimized nucleic acid sequence). For example, an exogenousnucleic acid can be a nucleic acid from one microorganism (e.g., onegenus or species of methylotrophic yeast) that is introduced into adifferent genus or species of methylotrophic yeast; however, anexogenous nucleic acid also can be a nucleic acid from a methylotrophicyeast that is introduced recombinantly into a methylotrophic yeast as anadditional copy despite the presence of a corresponding native nucleicacid sequence, or a nucleic acid from a methylotrophic yeast that isintroduced recombinantly into a methylotrophic yeast containing one ormore mutations, insertions, or deletions compared to the sequence nativeto the methylotrophic yeast. For example, P. pastoris contains anendogenous nucleic acid encoding an ALAS; an additional copy of the P.pastoris ALAS nucleic acid (e.g., introduced recombinantly into P.pastoris) is considered to be exogenous. Similarly, an “exogenous”protein is a protein encoded by an exogenous nucleic acid.

In some instances, an exogenous nucleic acid can be a heterologousnucleic acid. As used herein, a “heterologous” nucleic acid refers toany nucleic acid sequence that is not native to an organism (e.g., aheterologous nucleic acid can be a nucleic acid from one microorganism(e.g., one genus or species of methylotrophic yeast, whether or not ithas been codon-optimized) that is introduced into a different genus orspecies of methylotrophic yeast)). Similarly, a “heterologous” proteinis a protein encoded by a heterologous nucleic acid.

A nucleic acid molecule is considered to be exogenous to a host organismwhen any portion thereof (e.g., a promoter sequence or a sequence of anencoded protein) is exogenous to the host organism. A nucleic acidmolecule is considered to be heterologous to a host organism when anyportion thereof (e.g., a promoter sequence or a sequence of an encodedprotein) is heterologous to the host organism.

Nucleic acid constructs are provided herein that allow for geneticallyengineering a cell (e.g., a yeast cell (e.g., a methylotrophic yeastcell)). In some embodiments, nucleic acid constructs are provided hereinthat allow for genetically engineering a cell (e.g., a yeast cell (e.g.,a methylotrophic yeast cell)) to produce an RNA. Recombinantly producedRNAs can be used to modify a function of the cell, for example by RNAinterference or as a guide for DNA editing. In some embodiments, nucleicacid constructs are provided herein that allow for geneticallyengineering a cell (e.g., a yeast cell (e.g., a methylotrophic yeastcell)) to produce a product (e.g., a protein). In some embodiments,nucleic acid constructs are provided herein that allow for geneticallyengineering a cell (e.g., a yeast cell (e.g., a methylotrophic yeastcell)) to produce an exogenous product (e.g., a protein). In someembodiments, nucleic acid constructs are provided herein that allow forgenetically engineering a cell (e.g., a yeast cell (e.g., amethylotrophic yeast cell)) to produce a heterologous product (e.g., aprotein). In some embodiments, a nucleic acid constructs are providedherein that allow for genetically engineering a cell (e.g., a yeast cell(e.g., a methylotrophic yeast cell)) to produce a product (e.g., aprotein) in the absence of methanol. In addition, nucleic acidconstructs are provided herein that allow for genetically engineering acell (e.g., a yeast cell (e.g., a methylotrophic yeast cell)) toincrease the expression of a heme-binding protein.

Also provided herein is a cell including any of the promoter elementsdescribed herein. A cell can be any appropriate cell. For example, acell can be a bacterial cell (e.g., an E. coli cell, a B. subtilis cell,or a Lactococcus lactis cell), a fungal cell, an algal cell, a plantcell, an insect cell, or a mammalian cell. In some embodiments, a cellcan be a yeast cell. Non-limiting examples of yeast cells include Pichia(e.g., Pichia methanolica, Pichia pastoris), Candida (e.g., Candidaboidinii) cells, Hansenula (e.g., Hansenula polymorpha) cells,Torulopsis cells, and Sacharomyces (e.g., Sacharomyces cerevisae) cells.In some embodiments, a cell can be a methylotrophic yeast cell.Non-limiting examples of methylotrophic yeast cells include Pichiacells, Candida cells, Hansenula cells, and Torulopsis cells. In someembodiments, a cell can be a Pichia cell or a Sacharomyces cell.

In some embodiments, this document provides a cell containing a nucleicacid construct (e.g., a first nucleic acid construct, a second nucleicacid construct, and so forth) including a nucleotide sequence operablylinked to a promoter element as described herein. A nucleic acidconstruct including a nucleotide sequence can include any appropriatenucleotide sequence.

As used herein, “operably linked” means that a promoter or otherexpression element(s) are positioned relative to a coding sequence insuch a way as to direct or regulate expression of the coding sequence(e.g., in-frame).

It will be appreciated that a nucleic acid construct including anucleotide sequence operably linked to any of the promoter elements asdescribed herein can include nucleotide sequence of interest. In someembodiments, transcription and/or translation of a nucleotide sequencecan result in the production of a product (e.g., protein, DNA, RNA, or asmall molecule) of interest. For example, in some embodiments, a nucleicacid construct including a nucleotide sequence can be a nucleic acidconstruct encoding a protein. For example, in some embodiments, anucleic acid construct including a nucleotide sequence can be a nucleicacid construct encoding an RNA (e.g., an mRNA, a tRNA, a ribozyme, asiRNA, miRNA, or a shRNA). For example, in some embodiments, a nucleicacid construct including a nucleotide sequence can be a nucleic acidconstruct encoding a DNA. For example, in some embodiments, a nucleicacid construct including a nucleotide sequence can be a nucleic acidconstruct whose transcription results in or contributes to theproduction of a small molecule (e.g., heme, ethanol, or apharmaceutically active agent).

In some embodiments, a nucleic acid construct (e.g., a first nucleicacid construct, a second nucleic acid construct, and so forth) includinga nucleotide sequence can be a nucleic acid construct encoding a protein(e.g., a first protein, a second protein, and so forth).

Recombinantly expressed proteins can be widely used in manyapplications, such as for food, research, and medicine. In someembodiments, a protein encoded by a nucleic acid construct including anucleotide sequence operably linked to any of the promoter elements asdescribed herein can be a dehydrin, a phytase, a protease, a catalase, alipase, a peroxidase, an amylase, a transglutaminase, an oxidoreductase,a transferase, a hydrolase, a lyase, an isomerase, or a ligase. In someembodiments, a protein encoded by a nucleic acid operably linked to anyof the promoter elements as described herein can be an antibody orfragment thereof (e.g., adalimumab, rituximab, trastuzumab, bevacizumab,infliximab, or ranibizumab), an enzyme (e.g., a therapeutic enzyme suchas alpha-galactosidase A, alpha-L-iduronidase,N-acetylgalactosamine-4-sulfatase, dornase alfa, glucocerebrosidase,tissue plasminogen activator, rasburicase, an industrial enzyme (e.g., acatalase, a cellulase, a laccase, a glutaminase, or a glycosidase), or abiocatalyst (e.g., a transaminase, a cytochrome P450, a kinase, aphosphorylase, or an isomerase)), a regulatory protein (e.g., atranscription factor (e.g. Mxr1, Adr1)), a peptide hormone (e.g.,insulin, insulin-like growth factor 1, granulocyte colony-stimulatingfactor, follicle-stimulating hormone, or a growth hormone such as humangrowth hormone), a blood clotting protein (e.g., Factor VII), a cytokine(e.g., an interferon or erythropoietin), or a cytokine inhibitor (e.g.,etanercept).

In some embodiments, a protein can be a heme-binding protein (e.g., anexogenous or heterologous heme binding protein). In some embodiments, aheme-binding protein can be selected from the group consisting of aglobin (PF00042 in the Pfam database), a cytochrome (e.g., a cytochromeP450, a cytochrome a, a cytochrome b, a cytochrome c), a cytochrome coxidase, a ligninase, a catalase, and a peroxidase. In some embodiments,a globin can be selected from the group consisting of an androglobin, achlorocruorin, a cytoglobin, an erythrocruorin, a flavohemoglobin, aglobin E, a globin X, a globin Y, a hemoglobin (e.g., a beta hemoglobin,an alpha hemoglobin), a histoglobin, a leghemoglobin, a myoglobin, aneuroglobin, a non-symbiotic hemoglobin, a protoglobin, and a truncatedhemoglobin (e.g., a HbN, a HbO, a Glb3, a cyanoglobin). In someembodiments, the heme-binding protein can be a non-symbiotic hemoglobin.In some embodiments, the heme-binding protein can be a leghemoglobin. Insome embodiments, the heme-binding protein can be soybean leghemoglobin(LegH). A reference amino acid sequence for LegH is provided in FIG. 1as SEQ ID NO: 4. LegH is a protein that binds to heme, which results ina characteristic absorption at 415 nm and a distinct red color. The LegHprotein (also known as LGB2) is naturally found in root nodules ofsoybean (see, for example, UniprotKB Accession No. P02236). See, also,WO 2014/110539 and WO 2014/110532, each of which is incorporated byreference herein in its entirety. In some embodiments, a heme-bindingprotein can have an amino acid sequence that is at least 70% (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%) identical to the amino acid sequence set forth in any of SEQ IDNOs: 1-27 (FIG. 1). In some embodiments, a heme-binding protein is theamino acid sequence set forth in any of SEQ ID NOs: 1-27 (FIG. 1).

While the materials and methods are exemplified herein using an alcoholoxidase promoter element from a Pichia species (P. pastoris), otherorganisms can be used. For example, an alcohol oxidase promoter elementfrom a different methylotrophic yeast can be used, such as other speciesof the Pichia genus or species from any of the Candida, Hansenula,Pichia , and Torulopsis genera. Non-limiting examples of methylotrophicyeast species include Pichia methanolica, Pichia pastoris, Candidaboidinii, and Hansenula polymorpha (also called Pichia angusta). In someembodiments, a promoter element can be an alcohol oxidase promoterelement from any of the Candida, Hansenula, Pichia , and Torulopsisgenera. In some embodiments, a promoter element can have at least 70%(e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) sequenceidentity to an alcohol oxidase promoter element from any of the Candida,Hansenula, Pichia , and Torulopsis genera. In some embodiments, apromoter element can be an alcohol oxidase promoter element from any ofthe Candida, Hansenula, Pichia , and Torulopsis genera. In someembodiments, a promoter element can be an alcohol oxidase promoterelement from Pichia methanolica, Pichia pastoris, Candida boidinii, orHansenula polymorpha. In some embodiments, a promoter element can haveat least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%)sequence identity to an alcohol oxidase promoter element from Pichiamethanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha.In some embodiments, a promoter element can be an alcohol oxidasepromoter element from Pichia methanolica, Pichia pastoris, Candidaboidinii, or Hansenula polymorpha. Non-limiting examples of otheralcohol oxidase promoters include the AOX2 promoter from Pichiapastoris, pastoris (see, e.g., Ohi, Hideyuki, et al. Molecular andGeneral Genetics MGG 243.5 (1994): 489-499, incorporated herein byreference in its entirety), the alcohol oxidase (AOD1) promoter fromCandida boidinii (see, for example, GenBank Accession No. YSAAOD1A), thealcohol oxidase (MOX) promoter from Hansenula polymorpha (see, forexample, GenBank Accession No. X02425), or the MOD1 or MOD2 promoterfrom Pichia methanolica (see, for example, Raymond et al., 1998, Yeast,14:11-23; and Nakagawa et al., 1999, Yeast, 15:1223-30). In someembodiments, an alcohol oxidase promoter element can be selected fromthe group consisting of a promoter element from AOX1, AOX2, AOD1, MOX,MOD1, and MOD2. In some embodiments, an alcohol oxidase promoter elementcan be a promoter element from AOX1. In some embodiments, an alcoholoxidase promoter element can be a promoter element from AOX2. In someembodiments, an alcohol oxidase promoter element can be a promoterelement from AOD1. In some embodiments, an alcohol oxidase promoterelement can be a promoter element from MOX. In some embodiments, analcohol oxidase promoter element can be a promoter element from MOD1. Insome embodiments, an alcohol oxidase promoter element can be a promoterelement from MOD2.

In some embodiments, any of the cells (e.g., yeast cells (e.g.,methylotrophic yeast cells)) described herein can include a secondnucleic acid construct including a nucleotide sequence, transcriptionand/or translation of which can result in the production of a product asecond product (e.g., a protein, an RNA, a DNA, or a small molecule)operably linked to a promoter element. In some embodiments, the promoterelement to which the nucleotide sequence of the second nucleic acidconstruct is operably linked is the same as the promoter element towhich the nucleotide sequence of the first nucleic acid construct isoperably linked. In some embodiments, the promoter element to which thenucleotide sequence of the second nucleic acid construct is operablylinked is a second promoter element. In some embodiments, a secondpromoter element can be any of the promoter elements described herein.In some embodiments, the second promoter element can have the samesequence as the first promoter element. In some embodiments, the secondpromoter element can include one or more mutations corresponding tonucleotide positions 668-734 (e.g., nucleotide positions 673-729,nucleotide positions 678-724, nucleotide positions 683-719, ornucleotide positions 688-714) relative to SEQ ID NO: 28. In someembodiments, the second promoter element can include one or more (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19)mutations selected from the group consisting of mutations correspondingto T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C,T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862Arelative to SEQ ID NO: 28. In some embodiments, the second promoterelement can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, or 19) mutations selected from the groupconsisting of mutations corresponding to 146C, 154T, 303C, 426A, 433T,435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G,841T, and 862A relative to SEQ ID NO: 28, as long as the indicatednucleobase is not the same as the corresponding naturally-occurringnucleobase. In some embodiments, one or more (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations at positionscorresponding to the following positions relative to SEQ ID NO: 28 canbe present in a second promoter element: T146; C154; T303; T426; A433;A435; T530; C572; T596; T617; T688; A696; T702; A709; A712; T714; A790;A841; or T862. . In some embodiments, one or more (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations atpositions corresponding to the following positions relative to SEQ IDNO: 28 can be present in a second promoter element: 146; 154; 303; 426;433; 435; 530; 572; 596; 617; 688; 696; 702; 709; 712; 714; 790; 841; or862. In some embodiments, the second promoter element can include one ormore (e.g., 2, 3, 4, or 5) mutations selected from the group consistingof mutations corresponding to T688C, A696T, T702C, A712G, or T714Grelative to SEQ ID NO: 28. In some embodiments, the second promoterelement can include one or more (e.g., 2, 3, 4, or 5) mutations selectedfrom the group consisting of mutations corresponding to 688C, 696T,702C, 712G, or 714G relative to SEQ ID NO: 28, as long as the indicatednucleobase is not the same as the corresponding naturally-occurringnucleobase. In some embodiments, one or more (e.g., 2, 3, 4, or 5)mutations corresponding to one of the following positions relative toSEQ ID NO: 28 can be present in a second promoter element: T688; A696;T702; A712; or T714. In some embodiments, one or more (e.g., 2, 3, 4, or5) mutations corresponding to one of the following positions relative toSEQ ID NO: 28 can be present in a second promoter element: 688; 696;702; 712; or 714. In some embodiments, the second promoter element canbe an inducible promoter element (e.g., a methanol-inducible promoterelement) or a constitutive promoter element.

Any of a number of inducible promoters can generally be used whengenetically engineering cells (e.g., yeast (e.g., methylotrophic yeast).For example, a methanol-inducible promoter, or a promoter elementtherefrom, can be used. Suitable methanol inducible promoters includepAOX1, as described herein, as well as other methanol-induciblepromoters, or promoter elements therefrom. These include, withoutlimitation, the pAOX2 promoter from Pichia pastoris, the alcohol oxidase(AOD1) promoter from Candida boidinii (see, for example, GenBankAccession No. YSAAOD1A), the alcohol oxidase (MOX) promoter fromHansenula polymorpha (see, for example, GenBank Accession No. X02425),the MOD1 or MOD2 promoter from Pichia methanolica (see, for example,Raymond et al., 1998, Yeast, 14:11-23; and Nakagawa et al., 1999, Yeast,15:1223-30), the DHAS promoter from P. pastoris (see, for example,GenBank Accession No. FJ752551) or a promoter element therefrom, theformaldehyde dehydrogenase (FLD1) promoter from P. pastoris (see, forexample, GenBank Accession No. AF066054), or the PEX8 promoter from P.pastoris (see, for example, Kranthi et al., 2010, Yeast, 27:705-11). Allof these promoters are known to be induced by methanol. Suitableconstitutive promoters and constitutive promoter elements include,without limitation, the P. pastoris promoter (or a portion thereof) fromthe transcriptional elongation factor EF-1α gene (TEF1), which isstrongly transcribed in a constitutive manner. Other suitableconstitutive promoters (or promoter elements therefrom) also can beused, including, without limitation, the glyceraldehyde-3-phosphatedehydrogenase (GAPDH) promoter from P. pastoris (see, for example,GenBank Accession No. U62648.1), the promoter from the potentialglycosyl phosphatidyl inositol (GPI)-anchored protein, GCW14p(PAS_chr1-4_0586) from P. pastoris (see, for example, GenBank AccessionNo. XM_002490678), and the promoter from the 3-phosphoglycerate kinasegene (PGK1) from P. pastoris (see, for example, GenBank Accession No.AY288296). Further, it is noted that a combination of inducible (e.g.,methanol-inducible) and constitutive promoters (or promoter elementstherefrom) can be combined to further increase the expression of any ofthe nucleic acids operably linked thereto.

In some embodiments, a second protein can be any of the proteins asdescribed above. In some embodiments, the second protein can be atranscription factor (e.g., Mxr1). In some embodiments, any of thepromoter elements herein (e.g., a first promoter element or a secondpromoter element) can contain one or more recognition sequences for atranscription factor. Therefore, in some embodiments, a feedback loopmay be constructed such that the transcription factor drives theexpression of a protein of interest and also expression of additionalcopies of the transcription factor. In some embodiments, thetranscription factor can be Mxr1. In some embodiments, the secondprotein can be a protein involved in heme biosynthesis (e.g., a proteinselected from the group consisting of aminolevulinic acid synthase(ALAS), δ-aminolevulinic acid dehydratase (ALAD), porphobilinogendeaminase (PBGD), uroporphyrinogen III synthase (UPG3 S),uroporphyrinogen III decarboxylase (UPG3D), coprotoporphyrinogen oxidase(COPROX), protoporphyrinogen IX oxidase (PROTOX), and/or ferrochelatase(FC)).

Nucleic acids encoding one or more of the eight different enzymesinvolved in heme biosynthesis (as determined and annotated from thesequence of the Pichia pastoris genome) can be expressed as describedherein. For example, a heterologous nucleic acid molecule encoding ALAsynthase, ALA dehydratase, porphobilinogen deaminase, UPG III synthase,UPG III decarboxylase, CPG oxidase, PPG oxidase, and ferrochelatase canbe expressed in the strains (e.g., yeast strains (e.g., methylotrophicyeast strains)) described herein. For genetically engineering cells(e.g., yeast (e.g., methylotrophic yeast)) to contain more than oneheterologous nucleic acids (e.g., transgenes), a combination ofmethanol-inducible and constitutive promoters, or elements therefrom,can be combined to further increase the expression of such nucleicacids.

It will be understood that any of the cells (e.g., yeast cells (e.g.,methylotrophic yeast cells) described herein can include additionalnucleic acid constructs as a third, fourth, fifth, and so on, nucleicacid construct, and such constructs can, in some embodiments, be asdescribed above for a second nucleic acid construct.

Previous studies in Saccharomyces cerevisiae identified ALAD andporphobilinogen deaminase as rate limiting enzymes in heme biosynthesis(see, for example, Hoffman et al., 2003, Biochem. Biophys. Res. Commun.,310(4):1247-53). However, heterologous expression of individual hemeenzymes in P. pastoris from the glyceraldehyde-3-phosphate dehydrogenase(GAP) promoter failed to overcome limitations associated with theexpression of a recombinant protein containing a heme (see, Krainer etal., 2015, Microb. Cell Fact., 13;14:4). Expression of a recombinantheme containing protein in P. pastoris can be achieved by co-expressingthe entire heme biosynthetic pathway from methanol-inducible promoters,although it would be appreciated that one or more of the genes involvedin the heme biosynthetic pathway could be expressed from one or moreconstitutive promoters (see, e.g., U.S. Pat. No. 9,938,327, which isincorporated by reference in its entirety).

Also provided herein are methods of producing a product (e.g., aprotein) using any of the nucleic acid constructs and/or cells describedherein. In some embodiments, the methods provided herein can includeexpressing a nucleic acid construct including a nucleotide sequence(e.g., encoding a first protein) operably linked to a first promoterelement. In some embodiments, a first promoter element can be anypromoter element described herein. In some embodiments, the firstpromoter element includes one or more mutations corresponding tonucleotide positions 668-734 (e.g., nucleotide positions 673-729,nucleotide positions 678-724, nucleotide positions 683-719, ornucleotide positions 688-714) relative to SEQ ID NO: 28. In someembodiments, the methods provided herein can include expressing anucleic acid construct including a nucleotide sequence (e.g., encoding afirst protein) operably linked to a first promoter element, where thefirst promoter element includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations selected fromthe group consisting of mutations corresponding to T146C, C154T, T303C,T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C,A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28.. In some embodiments, the methods provided herein can includeexpressing a nucleic acid construct including a nucleotide sequence(e.g., encoding a first protein) operably linked to a first promoterelement, where the first promoter element includes one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19)mutations selected from the group consisting of mutations correspondingto 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C,696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ IDNO: 28, as long as the indicated nucleobase is not the same as thecorresponding naturally-occurring nucleobase. In some embodiments, themethods provided herein can include expressing a nucleic acid constructincluding a nucleotide sequence (e.g., encoding a first protein)operably linked to a first promoter element, wherein the promoterelement includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19) mutations at positions corresponding tothe following positions relative to SEQ ID NO: 28: T146; C154; T303;T426; A433; A435; T530; C572; T596; T617; T688; A696; T702; A709; A712;T714; A790; A841; or T862. In some embodiments, the methods providedherein can include expressing a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element, wherein the promoter element includes one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,or 19) mutations at positions corresponding to the following positionsrelative to SEQ ID NO: 28: 146; 154; 303; 426; 433; 435; 530; 572; 596;617; 688; 696; 702; 709; 712; 714; 790; 841; or 862. In someembodiments, the methods provided herein can include expressing anucleic acid construct including a nucleotide sequence (e.g., encoding afirst protein) operably linked to a first promoter element, where thefirst promoter element includes one or more (e.g., 2, 3, 4, or 5)mutations selected from the group consisting of mutations correspondingto T688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO: 28. Insome embodiments, the methods provided herein can include expressing anucleic acid construct including a nucleotide sequence (e.g., encoding afirst protein) operably linked to a first promoter element, where thefirst promoter element includes one or more (e.g., 2, 3, 4, or 5)mutations selected from the group consisting of mutations correspondingto 688C, 696T, 702C, 712G, and 714G relative to SEQ ID NO: 28, as longas the indicated nucleobase is not the same as the correspondingnaturally-occurring nucleobase. In some embodiments, the methodsprovided herein can include expressing a nucleic acid constructincluding a nucleotide sequence (e.g., encoding a first protein)operably linked to a first promoter element, wherein the promoterelement includes one or more one or more (e.g., 2, 3, 4, or 5) mutationsat positions corresponding to the following positions relative to SEQ IDNO: 28: T688; A696; T702; A712; or T714. In some embodiments, themethods provided herein can include expressing a nucleic acid constructincluding a nucleotide sequence (e.g., encoding a first protein)operably linked to a first promoter element, wherein the promoterelement includes one or more one or more (e.g., 2, 3, 4, or 5) mutationsat positions corresponding to the following positions relative to SEQ IDNO: 28: 688; 696; 702; 712; or 714. In some embodiments of any of themethods described herein, the method can be performed in the absence ofadded methanol. In some embodiments, the primary carbon source formethylotrophic yeast cells can be dextrose, sucrose, xylose, lactose,maltose, isomaltose, arabinose, sugar alcohols, ethanol, acetate, orglycerol. In some embodiments, the primary carbon source can be selectedfrom the group consisting of glucose, sucrose, sorbitol, methanol, andglycerol. In some embodiments, the primary carbon source can be selectedfrom the group consisting of glucose, sucrose, sorbitol, and glycerol.In some embodiments the primary carbon source can be an oligosaccharideor a polysaccharide (e.g., a starch, a pectin, cellulose, orhemicellulose). In some embodiments, the primary carbon source for amethylotrophic yeast cell can be a mixture of sugars (e.g., derived fromcellulosic biomass or starch).

In some embodiments, the methods provided herein allow for an increasein the titer of a product (e.g., a protein). In some embodiments, thetiter of a product (e.g., a protein) can be increased by at least 5%(e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%,800%, 900%, 1000%, or more) compared to a corresponding method lacking anucleic acid construct as described herein. In some embodiments, thetiter of a product (e.g., a protein) can be increased by at least 5%(e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%,800%, 900%, 1000% or more) compared to a corresponding method thatincludes expressing a nucleic acid encoding a first product (e.g., aprotein) operably linked to a first promoter element, where the firstpromoter element lacks any mutation in a nucleotide positioncorresponding to nucleotide positions 668-734 (e.g., nucleotidepositions 673-729, nucleotide positions 678-724, nucleotide positions683-719, or nucleotide positions 688-714) relative to SEQ ID NO: 28. Insome embodiments, the titer of a product (e.g., a protein) can beincreased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more) compared to acorresponding method that includes expressing a nucleic acid constructincluding a nucleotide sequence (e.g., encoding a first protein)operably linked to a first promoter element, where the first promoterelement lacks any mutation selected from the group consisting ofmutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G,T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G,A790G, A841T, and T862A relative to SEQ ID NO: 28. In some embodiments,the titer of a product (e.g., a protein) can be increased by at least 5%(e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%,800%, 900%, 1000% or more) compared to a corresponding method thatincludes expressing a nucleic acid construct including a nucleotidesequence (e.g., encoding a first protein) operably linked to a firstpromoter element, where the first promoter element lacks any mutationselected from the group consisting of mutations corresponding to 146C,154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C,709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ ID NO: 28, aslong as the indicated nucleobase is not the same as the correspondingnaturally-occurring nucleobase. In some embodiments, the titer of aproduct (e.g., a protein) can be increased by at least 5% (e.g., atleast 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%,1000% or more) compared to a corresponding method that includesexpressing a nucleic acid construct including a nucleotide sequence(e.g., encoding a first protein) operably linked to a first promoterelement, where the first promoter element lacks any mutation lacks anymutation in a nucleotide position corresponding to nucleotide positionsT146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696,T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO: 28.In some embodiments, the titer of a product (e.g., a protein) can beincreased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more) compared to acorresponding method that includes expressing a nucleic acid constructincluding a nucleotide sequence (e.g., encoding a first protein)operably linked to a first promoter element, where the first promoterelement lacks any mutation lacks any mutation in a nucleotide positioncorresponding to nucleotide positions 146, 154, 303, 426, 433, 435, 530,572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relativeto SEQ ID NO: 28.

In some embodiments, the titer of a product (e.g., a protein) can beincreased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more) compared to acorresponding method that includes expressing a nucleic acid constructincluding a nucleotide sequence (e.g., encoding a first protein)operably linked to a first promoter element, where the first promoterelement lacks any mutation selected from the group consisting ofmutations corresponding to T688C, A696T, T702C, A712G, and T714Grelative to SEQ ID NO: 28. In some embodiments, the titer of a product(e.g., a protein) can be increased by at least 5% (e.g., at least 6%,7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%or more) compared to a corresponding method that includes expressing anucleic acid construct including a nucleotide sequence (e.g., encoding afirst protein) operably linked to a first promoter element, where thefirst promoter element lacks any mutation selected from the groupconsisting of mutations corresponding to 688C, 696T, 702C, 712G, and714G relative to SEQ ID NO: 28. In some embodiments, the titer of aproduct (e.g., a protein) can be increased by at least 5% (e.g., atleast 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%,1000% or more) compared to a corresponding method that includesexpressing a nucleic acid construct including a nucleotide sequence(e.g., encoding a first protein) operably linked to a first promoterelement, where the first promoter element lacks any mutation lacks anymutation in a nucleotide position corresponding to nucleotide positionsT688, A696, T702, A712, and T714 relative to SEQ ID NO: 28. In someembodiments, the titer of a product (e.g., a protein) can be increasedby at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%,600%, 700%, 800%, 900%, 1000% or more) compared to a correspondingmethod that includes expressing a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element, where the first promoter element lacks anymutation lacks any mutation in a nucleotide position corresponding tonucleotide positions 688, 696, 702, 712, and 714 relative to SEQ ID NO:28.

Generally, a “titer” is the measurement of the amount of a substance insolution. As used herein, the “titer” of a heme-binding protein refersto the overall amount of the polypeptide, whether or not it is bound toheme, unless otherwise specified. The titer of a product (e.g., aprotein) can be measured by any suitable method, such ashigh-performance liquid chromatography (HPLC), high-performance liquidchromatography-mass spectrometry (HPLC-MS), enzyme-linked immunosorbentassay (ELISA), or ultraviolet and/or visible light spectroscopy.

As used herein, a “corresponding method” is a method that is essentiallyidentical to a reference method in all ways except for the identifieddifference. For example, a corresponding method for expressing a nucleicacid construct including a nucleotide sequence (e.g., encoding a firstprotein) operably linked to a first promoter element, where the firstpromoter element lacks one or more mutations corresponding to a mutationin a nucleotide position corresponding to nucleotide positions 668-734(e.g., nucleotide positions 673-729, nucleotide positions 678-724,nucleotide positions 683-719, or nucleotide positions 688-714) relativeto SEQ ID NO: 28 would essentially be the same as the reference methodin all aspects (e.g., genetic makeup of cell, temperature and time ofculture, and so forth), except that the corresponding method wouldexpress a nucleic acid construct including a nucleotide sequence (e.g.,encoding a first protein) operably linked to a first promoter elementthat lacks any mutations corresponding to a mutation in a nucleotideposition corresponding to nucleotide positions 668-734 (e.g., nucleotidepositions 673-729, nucleotide positions 678-724, nucleotide positions683-719, or nucleotide positions 688-714) relative to SEQ ID NO: 28. Forexample, a corresponding method for expressing a nucleic acid constructincluding a nucleotide sequence (e.g., encoding a first protein)operably linked to a first promoter element, where the first promoterelement lacks one or more mutations selected from the group consistingof mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G,T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G,A790G, A841T, and T862A relative to SEQ ID NO: 28 would essentially bethe same as the reference method in all aspects (e.g., genetic makeup ofcell, temperature and time of culture, and so forth), except that thecorresponding method would express a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element that lacks any of the mutations included in thegroup consisting of mutations corresponding to T146C, C154T, T303C,T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C,A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28.For example, a corresponding method for expressing a nucleic acidconstruct including a nucleotide sequence (e.g., encoding a firstprotein) operably linked to a first promoter element, where the firstpromoter element lacks one or more mutations at a nucleotide positioncorresponding to nucleotide positions T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO: 28 would essentially be the sameas the reference method in all aspects (e.g., genetic makeup of cell,temperature and time of culture, and so forth), except that thecorresponding method would express a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element that lacks any of the mutations at a nucleotideposition corresponding to nucleotide positions T146, C154, T303, T426,A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714,A790, A841, and T862 relative to SEQ ID NO: 28. For example, acorresponding method for expressing a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element, where the first promoter element lacks one ormore mutations selected from the group consisting of mutationscorresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C,617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A relativeto SEQ ID NO: 28 would essentially be the same as the reference methodin all aspects (e.g., genetic makeup of cell, temperature and time ofculture, and so forth), except that the corresponding method wouldexpress a nucleic acid construct including a nucleotide sequence (e.g.,encoding a first protein) operably linked to a first promoter elementthat lacks any of the mutations included in the group consisting ofmutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A,572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and862A relative to SEQ ID NO: 28. For example, a corresponding method forexpressing a nucleic acid construct including a nucleotide sequence(e.g., encoding a first protein) operably linked to a first promoterelement, where the first promoter element lacks one or more mutations ata nucleotide position corresponding to nucleotide positions 146, 154,303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709, 712, 714,790, 841, and 862 relative to SEQ ID NO: 28 would essentially be thesame as the reference method in all aspects (e.g., genetic makeup ofcell, temperature and time of culture, and so forth), except that thecorresponding method would express a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element that lacks any of the mutations at a nucleotideposition corresponding to nucleotide positions 146, 154, 303, 426, 433,435, 530, 572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862relative to SEQ ID NO: 28.

For example, a corresponding method for expressing a nucleic acidconstruct including a nucleotide sequence (e.g., encoding a firstprotein) operably linked to a first promoter element, where the firstpromoter element lacks one or more mutations selected from the groupconsisting of mutations corresponding to T688C, A696T, T702C, A712G, andT714G relative to SEQ ID NO: 28 would essentially be the same as thereference method in all aspects (e.g., genetic makeup of cell,temperature and time of culture, and so forth), except that thecorresponding method would express a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element that lacks any of the mutations included in thegroup consisting of mutations corresponding to T688C, A696T, T702C,A712G, and T714G relative to SEQ ID NO: 28. For example, a correspondingmethod for expressing a nucleic acid construct including a nucleotidesequence (e.g., encoding a first protein) operably linked to a firstpromoter element, where the first promoter element lacks one or moremutations selected from the group consisting of mutations correspondingto 688C, 696T, 702C, 712G, and 714G relative to SEQ ID NO: 28 wouldessentially be the same as the reference method in all aspects (e.g.,genetic makeup of cell, temperature and time of culture, and so forth),except that the corresponding method would express a nucleic acidconstruct including a nucleotide sequence (e.g., encoding a firstprotein) operably linked to a first promoter element that lacks any ofthe mutations included in the group consisting of mutationscorresponding to 688C, 696T, 702C, 712G, and 714G relative to SEQ ID NO:28. For example, a corresponding method for expressing a nucleic acidconstruct including a nucleotide sequence (e.g., encoding a firstprotein) operably linked to a first promoter element, where the firstpromoter element lacks one or more mutations at a nucleotide positioncorresponding to nucleotide positions T688, A696, T702, A712, and T714relative to SEQ ID NO: 28 would essentially be the same as the referencemethod in all aspects (e.g., genetic makeup of cell, temperature andtime of culture, and so forth), except that the corresponding methodwould express a nucleic acid construct including a nucleotide sequence(e.g., encoding a first protein) operably linked to a first promoterelement that lacks any of the mutations included in the group consistingof mutations corresponding to T688, A696, T702, A712, and T714 relativeto SEQ ID NO: 28. For example, a corresponding method for expressing anucleic acid construct including a nucleotide sequence (e.g., encoding afirst protein) operably linked to a first promoter element, where thefirst promoter element lacks one or more mutations at a nucleotideposition corresponding to nucleotide positions 688, 696, 702, 712, and714 relative to SEQ ID NO: 28 would essentially be the same as thereference method in all aspects (e.g., genetic makeup of cell,temperature and time of culture, and so forth), except that thecorresponding method would express a nucleic acid construct including anucleotide sequence (e.g., encoding a first protein) operably linked toa first promoter element that lacks any of the mutations included in thegroup consisting of mutations corresponding to 688, 696, 702, 712, and714 relative to SEQ ID NO: 28.

Genetically engineering a cell (e.g., a yeast cell (e.g., amethylotrophic yeast cell) typically includes introducing a recombinantnucleic acid molecule (also called a nucleic acid construct) into thecell. As described herein, a recombinant nucleic acid molecule typicallyincludes an exogenous nucleic acid that encodes a product (e.g., aprotein (e.g., a protein involved in heme biosynthesis, a heme-bindingprotein, or a transcription factor)) operably linked to at least onepromoter element (e.g., an inducible or constitutive promoter element).In some embodiments, a recombinant nucleic acid molecule can include alinear sequence of two or more protein-coding sequences operably linkedto the same or separate promoter elements (e.g., a first promoteroperably linked to a first nucleic acid construct including a nucleotidesequence (e.g., encoding a first protein) and a second promoter operablylinked to a second nucleic acid construct including a nucleotidesequence (e.g., encoding a second protein), or a promoter operablylinked to a first nucleic acid construct including a nucleotide sequence(e.g., encoding a first protein) and a second nucleic acid constructincluding a nucleotide sequence (e.g., encoding a second protein)). Insome cases, a recombinant nucleic acid molecule including at least onepromoter operably linked to a nucleotide sequence (e.g., encoding aprotein) can be called a cassette.

A recombinant nucleic acid can include expression elements. Expressionelements include nucleic acid sequences that direct and regulateexpression of nucleic acid coding sequences. One example of anexpression element is a promoter sequence. Expression elements also caninclude introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin, andvectors can contain a combination of elements from different origins.

Nucleic acids can be detected using any number of amplificationtechniques (see, e.g., PCR Primer: A Laboratory Manual, 1995,Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188) with an appropriate pair of oligonucleotides (e.g.,primers). A number of modifications to the original PCR method have beendeveloped and can be used to detect selected nucleic acids.

Suitable transcription factors, and nucleic acids encoding transcriptionfactors (e.g., exogenous nucleic acids encoding transcription factors)include, for example, Mxr1 from Pichia pastoris. A representative P.pastoris Mxr1nucleic acid sequence can be found, for example, in GenBankAccession No. DQ395124, while a representative P. pastorisMxr1polypeptide sequence can be found, for example, in GenBank AccessionNo. ABD57365.). In some embodiments, the transcription factor can be aMitl sequence from P. pastoris (see, for example, GenBank Accession No.CAY70887). Suitable transcription factors also can be found in Hansenulapolymorpha (e.g., Adr1; see, for example, GenBank Accession No.AEOI02000005, bases 858873 to 862352, for the nucleic acid sequence andGenBank Accession No. ESX01253 for the amino acid sequence) and Candidaboidinii (e.g., Trm1; see, for example, GenBank Accession No. AB365355for the nucleic acid sequence and GenBank Accession No. BAF99700 for theamino acid sequence; and Trm2; see, for example, GenBank Accession No.AB548760 for the nucleic acid sequence and GenBank Accession No.BAJ07608 for the amino acid sequence).

Transcription factors such as Mxr1 may be normally expressed at lowlevels. In some embodiments, it is desirable to place the exogenousnucleic acid (e.g., the transcription factor) under control of apromoter that is inducible.

In some embodiments, a transcription factor can bind to a promoterelement as described herein and activate transcription from the promoterelement. In some embodiments, when a nucleic acid sequence encoding thetranscription factor is operably linked to a promoter element to whichit binds, a positive feedback loop can be created to help driveexpression of other nucleic acid sequences (e.g., protein-encodingnucleic acid sequences) operably linked to the promoter. Non-limitingexamples of transcription factors that can be used with an AOX1 promoter(e.g., a mutated AOX1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2,and combinations thereof In some embodiments, a transcription factorthat can be used with an AOX1 promoter can include Mxr1. A non-limitingexample of a transcription factor that can be used with an MOX promoter(e.g., a mutated MOX promoter) is Adr1. Non-limiting examples oftranscription factors that can be used with an AOD1 promoter (e.g., amutated AOD1 promoter) include Trm1, Trm2, or a combination thereof Insome embodiments, two methanol-regulated transcription factors (e.g.,Mxr1 and Mit1) can be operably linked to a methanol inducible promoterelement (e.g., pAOX1).

The recombinant nucleic acid molecules described herein can be stablyintegrated into the genome of the cell (e.g., yeast cell (e.g.,methylotrophic yeast cell), or can be extrachromosomally expressed froma replication-competent plasmid. Methods of achieving both are wellknown and routinely used in the art.

In addition, it is noted that a first nucleic acid construct including anucleotide sequence (e.g., encoding a first protein (e.g., aheme-binding protein)) operably linked to a promoter element (e.g., apromoter element as described herein) can be physically separate from asecond nucleic acid construct including a nucleotide sequence (e.g.,encoding a second protein (e.g., a transcription factor)) operablylinked to a promoter element (e.g., a promoter element as describedherein) (that is, the first and second nucleic acid constructs can becompletely separate molecules). Alternatively, a first nucleic acidconstruct including a nucleotide sequence (e.g., encoding a firstprotein) operably linked to a promoter element (e.g., a promoter elementas described herein) and a second nucleic acid construct including anucleotide sequence (e.g., encoding a second protein) operably linked toa promoter element (e.g., a promoter element as described herein) can beincluded in the same nucleic acid construct. In some embodiments, afirst nucleic acid construct including a nucleotide sequence (e.g.,encoding a first protein) operably linked to a promoter element can becontiguous with a second nucleic acid construct including a nucleotidesequence (e.g., encoding a second protein) operably linked to a promoterelement. It would be appreciated by a skilled artisan that, if thesecond nucleic acid construct including a nucleotide sequence (e.g.,encoding a second protein) is contiguous with the first nucleic acidconstruct including a nucleotide sequence (e.g., encoding a protein ofinterest), a single promoter, or promoter element therefrom, can be usedto drive transcription of both or all of the nucleotide sequences (e.g.,a nucleic acid encoding the first protein as well as a second protein).

Methods of introducing nucleic acids into cells (e.g., yeast cells(e.g., methylotrophic yeast cells)) are known in the art, and include,without limitation, transduction, electroporation, biolistic particledelivery, and chemical transformation. Methods of culturing cells (e.g.,yeast cells (e.g., methylotrophic yeast cells)) also are known in theart. See, for example, Pichia Protocols, Methods In Molecular Biology,389, Cregg, Ed., 2007, 2^(nd) Ed., Humana Press, Inc. Under somecircumstances, it may be desirable to introduce or add methanol to theculture media, although, as demonstrated herein, methanol is notrequired to obtain efficient expression at high levels of one or moreproducts (e.g., proteins) of interest. Under some circumstances (e.g.,when one or more nucleic acids encoding enzyme(s) involved in hemebiosynthesis are expressed), it may be desirable to supplement theculture media with iron or a pharmaceutically or metabolicallyacceptable (or GRAS) salt thereof.

The methods provided herein also can include purifying an expressedprotein. As used herein, an “enriched” protein is a protein thataccounts for at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more) by dry weight, of themass of the production cell, or at least 10% (e.g., at least 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or99%) by dry weight, the mass of the production cell lysate (e.g.,excluding cell wall or membrane material). As used herein, a “purified”protein is a protein that has been separated from cellular componentsthat naturally accompany it. Typically, the protein is considered“purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%,95%, or 99%) by dry weight, free from other proteins and naturallyoccurring molecules with which it is naturally associated.

As used herein, nucleic acids can include DNA and RNA, and includesnucleic acids that contain one or more nucleotide analogs or backbonemodifications. A nucleic acid can be single stranded or double stranded,which usually depends upon its intended use. Also provided are nucleicacids and polypeptides that differ from a given sequence. Nucleic acidsand polypeptides can have at least 50% sequence identity (e.g., at least55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to a given nucleic acid or polypeptide sequence. In someembodiments, a nucleic acid or polypeptide can have 100% sequenceidentity to a given nucleic acid or polypeptide sequence.

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of nucleotides or amino acid residuesbetween the two sequences is determined. The number of identical matchesis divided by the length of the aligned region (i.e., the number ofaligned nucleotides or amino acid residues) and multiplied by 100 toarrive at a percent sequence identity value. It will be appreciated thatthe length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. It alsowill be appreciated that a single sequence can align with more than oneother sequence and hence, can have different percent sequence identityvalues over each aligned region.

The alignment of two or more sequences to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid orpolypeptide sequences to be carried out across their entire length(global alignment). Chenna et al., 2003, Nucleic Acids Res.,31(13):3497-500. ClustalW calculates the best match between a query andone or more subject sequences, and aligns them so that identities,similarities and differences can be determined. Gaps of one or moreresidues can be inserted into a query sequence, a subject sequence, orboth, to maximize sequence alignments. For fast pairwise alignment ofnucleic acid sequences, the default parameters can be used (i.e., wordsize: 2; window size: 4; scoring method: percentage; number of topdiagonals: 4; and gap penalty: 5); for an alignment of multiple nucleicacid sequences, the following parameters can be used: gap openingpenalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes.For fast pairwise alignment of polypeptide sequences, the followingparameters can be used: word size: 1; window size: 5; scoring method:percentage; number of top diagonals: 5; and gap penalty: 3. For multiplealignment of polypeptide sequences, the following parameters can beused: weight matrix: blosum; gap opening penalty: 10.0; gap extensionpenalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro,Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gappenalties: on. ClustalW can be run, for example, at the Baylor Collegeof Medicine Search Launcher website or at the European BioinformaticsInstitute website on the World Wide Web.

Changes can be introduced into a nucleic acid molecule, thereby leadingto changes in the amino acid sequence of the encoded polypeptide. Forexample, changes can be introduced into nucleic acid coding sequencesusing mutagenesis (e.g., site-directed mutagenesis, PCR-mediatedmutagenesis, transposon mutagenesis, chemical mutagenesis, UVmutagenesis or radiation induced mutagenesis) or by chemicallysynthesizing a nucleic acid molecule having such changes. Such nucleicacid changes can lead to conservative and/or non-conservative amino acidsubstitutions at one or more amino acid residues. A “conservative aminoacid substitution” is one in which one amino acid residue is replacedwith a different amino acid residue having a similar side chain (see,for example, Dayhoff et al., 1978, Atlas of Protein Sequence andStructure, 5(Suppl. 3):345-352, which provides frequency tables foramino acid substitutions), and a non-conservative substitution is one inwhich an amino acid residue is replaced with an amino acid residue thatdoes not have a similar side chain. Nucleic acid and/or polypeptidesequences may be modified as described herein to improve one or moreproperties such as, without limitation, increased expression (e.g.,transcription and/or translation), tighter regulation, deregulation,loss of catabolite repression, modified specificity, secretion,thermostability, solvent stability, oxidative stability, proteaseresistance, catalytic activity, and/or color.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is free of sequences that naturally flank one or both endsof the nucleic acid in the genome of the organism from which theisolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNAfragment produced by PCR or restriction endonuclease digestion). Such anisolated nucleic acid molecule is generally introduced into a vector(e.g., a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule, discussed inmore detail below. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule.

Vectors as described herein can be introduced into a host cell. As usedherein, “host cell” refers to the particular cell into which the nucleicacid is introduced and also includes the progeny of such a cell thatcarry the vector. A host cell can be any prokaryotic or eukaryotic cell.For example, nucleic acids can be expressed in bacterial cells such asE. coli, or in insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art. Many methods for introducing nucleicacids into host cells, both in vivo and in vitro, are well known tothose skilled in the art and include, without limitation,electroporation, calcium phosphate precipitation, polyethylene glycol(PEG) transformation, heat shock, lipofection, microinjection, andviral-mediated nucleic acid transfer.

Nucleic acids can be isolated using techniques routine in the art. Forexample, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides.

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A polypeptide also can be purified, forexample, by expressing a nucleic acid in an expression vector. Inaddition, a purified polypeptide can be obtained by chemical synthesis.The extent of purity of a polypeptide can be measured using anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A construct or vector containing a nucleic acid construct as describedherein (e.g., a nucleotide sequence that encodes a polypeptide operablylinked to a promoter element as described herein) also is provided.Constructs or vectors, including expression constructs or vectors, arecommercially available or can be produced by recombinant DNA techniquesroutine in the art. A construct or vector containing a nucleic acid canhave expression elements operably linked to such a nucleic acid, andfurther can include sequences such as those encoding a selectable marker(e.g., an antibiotic resistance gene). A construct or vector containinga nucleic acid can encode a chimeric or fusion polypeptide (i.e., apolypeptide operatively linked to a heterologous polypeptide, which canbe at either the N-terminus or C-terminus of the polypeptide).Representative heterologous polypeptides are those that can be used inpurification of the encoded polypeptide (e.g., 6× His tag, glutathioneS-transferase (GST)).

Nucleic acids also can be detected using hybridization. Hybridizationbetween nucleic acids is discussed in detail in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sections 7.37-7.57,9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. disclosessuitable Southern blot conditions for oligonucleotide probes less thanabout 100 nucleotides (Sections 11.45-11.46). The Tm between a sequencethat is less than 100 nucleotides in length and a second sequence can becalculated using the formula provided in Section 11.46. Sambrook et al.additionally discloses Southern blot conditions for oligonucleotideprobes greater than about 100 nucleotides (see Sections 9.47-9.54). TheTm between a sequence greater than 100 nucleotides in length and asecond sequence can be calculated using the formula provided in Sections9.50-9.51 of Sambrook et al.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe, can play a significant role in thestringency of the hybridization. Such hybridizations and washes can beperformed, where appropriate, under moderate or high stringencyconditions. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Simply byway of example, high stringency conditions typically include a wash ofthe membranes in 0.2× SSC at 65° C.

In addition, interpreting the amount of hybridization can be affected,for example, by the specific activity of the labeled oligonucleotideprobe, by the number of probe-binding sites on the template nucleic acidto which the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium. It will be readily appreciatedby those of ordinary skill in the art that although any number ofhybridization and washing conditions can be used to examinehybridization of a probe nucleic acid molecule to immobilized targetnucleic acids, it is more important to examine hybridization of a probeto target nucleic acids under identical hybridization, washing, andexposure conditions. Preferably, the target nucleic acids are on thesame membrane.

A nucleic acid molecule is deemed to hybridize to a nucleic acid but notto another nucleic acid if hybridization to a nucleic acid is at least5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,50-fold, or 100-fold) greater than hybridization to another nucleicacid. The amount of hybridization can be quantitated directly on amembrane or from an autoradiograph using, for example, a Phosphorlmageror a Densitometer (Molecular Dynamics, Sunnyvale, Calif.).

Polypeptides can be detected using antibodies. Techniques for detectingpolypeptides using antibodies include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Anantibody can be polyclonal or monoclonal. An antibody having specificbinding affinity for a polypeptide can be generated using methods wellknown in the art. The antibody can be attached to a solid support suchas a microtiter plate using methods known in the art. In the presence ofa polypeptide, an antibody-polypeptide complex is formed.

Detection (e.g., of an amplification product, a hybridization complex,or a polypeptide) is usually accomplished using detectable labels. Theterm “label” is intended to encompass the use of direct labels as wellas indirect labels. Detectable labels include enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials.

Methods are described herein that can be used to generate a strain thatlacks sequences for selection (i.e., that lacks a selectable marker).These methods include using a circular plasmid DNA vector and a linearDNA sequence; the circular plasmid DNA vector contains a selectionmarker and an origin of DNA replication (also known as an autonomouslyreplicating sequence (ARS)), and the linear DNA sequence containssequences for integration into the Pichia genome by homologousrecombination. A linear DNA molecule additionally can include nucleicacid sequences encoding one or more proteins of interest such as,without limitation, heme-bound LegH, a dehydrin, a phytase, a protease acatalase, a lipase, a peroxidase, an amylase, a transglutaminase, anoxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, aligase, one or more enzymes involved in the pathway for production ofsmall molecules, such as ethanol, lactic acid, butanol, adipic acid orsuccinic acid, or an antibody against any such proteins.

Cells (e.g., yeast cells (e.g., methylotrophic yeast cells (e.g.,Pichia))) can be transformed with both DNA molecules and thetransformants selected by the presence of the selectable marker on thecircular plasmid. Transformants then can be screened for integration ofthe linear DNA molecule into the genome using, for example, PCR. Oncetransformants with the correct integration of the marker-free linear DNAmolecule are identified, the cells can be grown in the absence ofselection for the circular plasmid. Because the marker-bearing plasmidis not stably maintained in the absence of selection, the plasmid islost, often very quickly, after selection is relaxed. The resultingstrain carries the integrated linear DNA in the absence of heterologoussequences for selection. Therefore, this approach can be used toconstruct strains (e.g., Pichia strains) that lack a selectable marker(e.g., a heterologous selection marker) with little to no impact onrecombinant product (e.g., protein) yield.

In accordance with the present disclosure, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The materials and methods of thedisclosure will be further described in the following examples, which donot limit the scope of the methods and compositions of matter describedin the claims.

Exemplary Embodiments

Embodiment 1 is a nucleic acid construct comprising a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises a mutation at one or more nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO: 28.

Embodiment 2 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions673-729 relative to SEQ ID NO: 28.

Embodiment 3 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions678-724 relative to SEQ ID NO: 28.

Embodiment 4 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions683-719 relative to SEQ ID NO: 28.

Embodiment 5 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions688-714 relative to SEQ ID NO: 28.

Embodiment 6 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises two or more mutationsat nucleotide positions corresponding to any of nucleotide positions668-734 relative to SEQ ID NO: 28.

Embodiment 7 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises three or more mutationsat nucleotide positions corresponding to any of nucleotide positions668-734 relative to SEQ ID NO: 28.

Embodiment 8 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises four or more mutationsat nucleotide positions corresponding to any of nucleotide positions668-734 relative to SEQ ID NO: 28.

Embodiment 9 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter element comprises five or more mutationsat nucleotide positions corresponding to any of nucleotide positions668-734 relative to SEQ ID NO: 28.

Embodiment 10 is the nucleic acid construct of embodiment 1, wherein thefirst alcohol oxidase promoter has the sequence of SEQ ID NO: 29.

Embodiment 11 is a nucleic acid construct comprising a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises one or more mutations at a nucleotide positionselected from the group consisting of nucleotide positions correspondingto T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688,A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ IDNO: 28.

Embodiment 12 is the nucleic acid construct of embodiment 11, whereinthe first alcohol oxidase promoter element comprises two or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO: 28.

Embodiment 13 is the nucleic acid construct of embodiment 11, whereinthe first alcohol oxidase promoter element comprises three or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO: 28.

Embodiment 14 is the nucleic acid construct of embodiment 11, whereinthe first alcohol oxidase promoter element comprises four or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO: 28.

Embodiment 15 is the nucleic acid construct of embodiment 11, whereinthe first alcohol oxidase promoter element comprises five or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO: 28.

Embodiment 16 is the nucleic acid construct of any one of embodiments11-15, wherein the first alcohol oxidase promoter element comprises oneor more mutations at a nucleotide position selected from the groupconsisting of nucleotide positions corresponding to T688, A696, T702,A712, and T714 relative to SEQ ID NO: 28.

Embodiment 17 is the nucleic acid construct of any one of embodiments11-15, wherein the first alcohol oxidase promoter element comprises twoor more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to T688, A696, T702,A712, and T714 relative to SEQ ID NO: 28.

Embodiment 18 is the nucleic acid construct of any one of embodiments11-15, wherein the first alcohol oxidase promoter element comprisesthree or more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to T688, A696, T702,A712, and T714 relative to SEQ ID NO: 28.

Embodiment 19 is the nucleic acid construct of any one of embodiments11-15, wherein the first alcohol oxidase promoter element comprises fouror more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to T688, A696, T702,A712, and T714 relative to SEQ ID NO: 28.

Embodiment 20 is the nucleic acid construct of any one of embodiments11-15, wherein the first alcohol oxidase promoter element comprisesmutations at nucleotide positions corresponding to T688, A696, T702,A712, and T714 relative to SEQ ID NO: 28.

Embodiment 21 is a nucleic acid construct comprising a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises one or more mutations at a nucleotide positionselected from the group consisting of nucleotide positions correspondingto 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709,712, 714, 790, 841, and 862 relative to SEQ ID NO: 28.

Embodiment 22 is the nucleic acid construct of embodiment 21, whereinthe first alcohol oxidase promoter element comprises two or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530,572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relativeto SEQ ID NO: 28.

Embodiment 23 is the nucleic acid construct of embodiment 21, whereinthe first alcohol oxidase promoter element comprises three or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530,572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relativeto SEQ ID NO: 28.

Embodiment 24 is the nucleic acid construct of embodiment 21, whereinthe first alcohol oxidase promoter element comprises four or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530,572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relativeto SEQ ID NO: 28.

Embodiment 25 is the nucleic acid construct of embodiment 21, whereinthe first alcohol oxidase promoter element comprises five or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530,572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relativeto SEQ ID NO: 28.

Embodiment 26 is the nucleic acid construct of any one of embodiments21-25, wherein the first alcohol oxidase promoter element comprises oneor more mutations at a nucleotide position selected from the groupconsisting of nucleotide positions corresponding to 688, 696, 702, 712,and 714 relative to SEQ ID NO: 28.

Embodiment 27 is the nucleic acid construct of any one of embodiments21-25, wherein the first alcohol oxidase promoter element comprises twoor more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to 688, 696, 702, 712,and 714 relative to SEQ ID NO: 28.

Embodiment 28 is the nucleic acid construct of any one of embodiments21-25, wherein the first alcohol oxidase promoter element comprisesthree or more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to 688, 696, 702, 712,and 714 relative to SEQ ID NO: 28.

Embodiment 29 is the nucleic acid construct of any one of embodiments21-25, wherein the first alcohol oxidase promoter element comprises fouror more mutations at nucleotide positions selected from the groupconsisting of nucleotide positions corresponding to 688, 696, 702, 712,and 714 relative to SEQ ID NO: 28.

Embodiment 30 is the nucleic acid construct of any one of embodiments21-25, wherein the first alcohol oxidase promoter element comprisesmutations at nucleotide positions corresponding to 688, 696, 702, 712,and 714 relative to SEQ ID NO: 28.

Embodiment 31 is a nucleic acid construct comprising a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises one or more mutations selected from the groupconsisting of mutations corresponding to T146C, C154T, T303C, T426A,A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G,A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28.

Embodiment 32 is the nucleic acid construct of embodiment 31, whereinthe first alcohol oxidase promoter element comprises two or moremutations selected from the group consisting of mutations correspondingto T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C,T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862Arelative to SEQ ID NO: 28.

Embodiment 33 is the nucleic acid construct of embodiment 31, whereinthe first alcohol oxidase promoter element comprises three or moremutations selected from the group consisting of mutations correspondingto T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C,T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862Arelative to SEQ ID NO: 28.

Embodiment 34 is the nucleic acid construct of embodiment 31, whereinthe first alcohol oxidase promoter element comprises four or moremutations selected from the group consisting of mutations correspondingto T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C,T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862Arelative to SEQ ID NO: 28.

Embodiment 35 is the nucleic acid construct of embodiment 31, whereinthe first alcohol oxidase promoter element comprises five or moremutations selected from the group consisting of mutations correspondingto T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C,T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862Arelative to SEQ ID NO: 28.

Embodiment 36 is the nucleic acid construct of any one of embodiments1-35, wherein the first alcohol oxidase promoter element comprises oneor more mutations selected from the group consisting of T688C, A696T,T702C, A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 37 is the nucleic acid construct of any one of embodiments1-35, wherein the first alcohol oxidase promoter element comprises twoor more mutations selected from the group consisting of T688C, A696T,T702C, A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 38 is the nucleic acid construct of any one of embodiments1-35, wherein the first alcohol oxidase promoter element comprises threeor more mutations selected from the group consisting of T688C, A696T,T702C, A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 39 is the nucleic acid construct of any one of embodiments1-35, wherein the first alcohol oxidase promoter element comprises fouror more mutations selected from the group consisting of T688C, A696T,T702C, A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 40 is the nucleic acid construct of any one of embodiments1-35, wherein the first alcohol oxidase promoter element comprises themutations T688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO:28.

Embodiment 41 is a nucleic acid construct comprising a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises one or more mutations selected from the groupconsisting of mutations corresponding to 146C, 154T, 303C, 426A, 433T,435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G,841T, and 862A relative to SEQ ID NO: 28.

Embodiment 42 is the nucleic acid construct of embodiment 41, whereinthe first alcohol oxidase promoter element comprises two or moremutations selected from the group consisting of mutations correspondingto 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C,696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ IDNO: 28.

Embodiment 43 is the nucleic acid construct of embodiment 41, whereinthe first alcohol oxidase promoter element comprises three or moremutations selected from the group consisting of mutations correspondingto 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C,696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ IDNO: 28.

Embodiment 44 is the nucleic acid construct of embodiment 41, whereinthe first alcohol oxidase promoter element comprises four or moremutations selected from the group consisting of mutations correspondingto 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C,696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ IDNO: 28.

Embodiment 45 is the nucleic acid construct of embodiment 41, whereinthe first alcohol oxidase promoter element comprises five or moremutations selected from the group consisting of mutations correspondingto 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C,696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ IDNO: 28.

Embodiment 46 is the nucleic acid construct of any one of embodiments1-45, wherein the first alcohol oxidase promoter element comprises oneor more mutations selected from the group consisting of 688C, 696T,702C, 712G, and 714G relative to SEQ ID NO: 28.

Embodiment 47 is the nucleic acid construct of any one of embodiments1-45, wherein the first alcohol oxidase promoter element comprises twoor more mutations selected from the group consisting of 688C, 696T,702C, 712G, and 714G relative to SEQ ID NO: 28.

Embodiment 48 is the nucleic acid construct of any one of embodiments1-45, wherein the first alcohol oxidase promoter element comprises threeor more mutations selected from the group consisting of 688C, 696T,702C, 712G, and 714G relative to SEQ ID NO: 28.

Embodiment 49 is the nucleic acid construct of any one of embodiments1-45, wherein the first alcohol oxidase promoter element comprises fouror more mutations selected from the group consisting of 688C, 696T,702C, 712G, and 714G relative to SEQ ID NO: 28.

Embodiment 50 is the nucleic acid construct of any one of embodiments1-45, wherein the first alcohol oxidase promoter element comprises themutations 688C, 696T, 702C, 712G, and 714G relative to SEQ ID NO: 28.

Embodiment 51 is the nucleic acid construct of any one of embodiments1-50, wherein the first alcohol oxidase promoter element is an alcoholoxidase promoter element from a promoter selected from the groupconsisting of AOX1, AOX2, AOD1, MOX, MOD1, and MOD2.

Embodiment 52 is the nucleic acid construct of any one of embodiments1-51, wherein the first alcohol oxidase promoter element is an alcoholoxidase 1 (AOX1) promoter element.

Embodiment 53 is the nucleic acid construct of any one of embodiments1-52, wherein the first alcohol oxidase promoter element has at least90% sequence identity to SEQ ID NO: 28.

Embodiment 54 is the nucleic acid construct of any one of embodiments1-52, wherein the first alcohol oxidase promoter element has at least95% sequence identity to SEQ ID NO: 28.

Embodiment 55 is the nucleic acid construct of any one of embodiments1-54, further comprising a nucleotide sequence, wherein the nucleotidesequence is operably linked to the first alcohol oxidase promoterelement.

Embodiment 56 is the nucleic acid construct of embodiment 55, whereinthe nucleotide sequence encodes a first protein.

Embodiment 57 is the nucleic acid construct of embodiment 56, whereinthe first protein is exogenous to a methylotrophic yeast cell.

Embodiment 58 is the nucleic acid construct of embodiment 56 orembodiment 57, wherein the first protein is heterologous to amethylotrophic yeast cell.

Embodiment 59 is the nucleic acid construct of any one of embodiments56-58, wherein the first protein is selected from the group consistingof an antibody or fragment thereof, an enzyme, a regulatory protein, apeptide hormone, a blood clotting protein, a cytokine, a cytokineinhibitor, and a heme-binding protein.

Embodiment 60 is the nucleic acid construct of any one of embodiments56-59, wherein the first protein is a heme-binding protein.

Embodiment 61 is the nucleic acid construct of embodiment 60, whereinthe heme-binding protein is selected from the group consisting of aglobin, a cytochrome, a cytochrome c oxidase, a ligninase, a catalase,and a peroxidase.

Embodiment 62 is the nucleic acid construct of embodiment 60, whereinthe heme-binding protein is selected from the group consisting of anandroglobin, a chlorocruorin, a cytoglobin, an erythrocruorin, aflavohemoglobin, a globin E, a globin X, a globin Y, a hemoglobin, ahistoglobin, a leghemoglobin, a myoglobin, a neuroglobin, anon-symbiotic hemoglobin, a protoglobin, and a truncated hemoglobin.

Embodiment 63 is the nucleic acid construct of embodiment 60, whereinthe heme-binding protein is a non-symbiotic hemoglobin.

Embodiment 64 is the nucleic acid construct of embodiment 60, whereinthe heme-binding protein is a leghemoglobin.

Embodiment 65 is the nucleic acid construct of embodiment 60, whereinthe heme-binding protein comprises an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of any of SEQ IDNOs: 1-27.

Embodiment 66 is the nucleic acid construct of any one of embodiments1-65, wherein the first alcohol oxidase promoter element comprises arecognition sequence for a transcription factor.

Embodiment 67 is a cell comprising a first nucleic acid construct,wherein the first nucleic acid construct is the nucleic acid constructof any one of embodiments 1-66.

Embodiment 68 is the cell of embodiment 67, wherein the cell is a yeastcell.

Embodiment 69 is the cell of embodiment 68, wherein the yeast cell is amethylotrophic yeast cell.

Embodiment 70 is the cell of embodiment 69, wherein the methylotrophicyeast cell is a Pichia cell, a Candida cell, a Hansenula cell, or aTorulopsis cell.

Embodiment 71 is the cell of embodiment 69 or embodiment 70, wherein themethylotrophic yeast cell is a Pichia methanolica cell, a Pichiapastoris cell, a Candida boidinii cell, or a Hansenula polymorpha cell.

Embodiment 72 is the cell of any one of embodiments 69-71, wherein themethylotrophic yeast cell is a Pichia pastoris cell.

Embodiment 73 is the cell of any one of embodiments 67-72, furthercomprising a second nucleic acid construct comprising a nucleotidesequence, wherein the nucleotide sequence is operably linked to thefirst alcohol oxidase promoter element or to a second promoter element.

Embodiment 74 is the cell of embodiment 73, wherein the nucleotidesequence of the second nucleic acid construct is operably linked to asecond promoter element that has the same sequence as the first alcoholoxidase promoter element.

Embodiment 75 is the cell of any one of embodiments 73-74, wherein thenucleotide sequence of the second nucleic acid construct encodes asecond protein.

Embodiment 76 is the cell of embodiment 75, wherein the second proteinis a transcription factor.

Embodiment 77 is the cell of embodiment 76, wherein the nucleotidesequence encoding the second protein is operably linked to a secondpromoter element that comprises a recognition sequence for thetranscription factor.

Embodiment 78 is the cell of embodiment 76 or embodiment 77, wherein thefirst alcohol oxidase promoter element comprises a recognition sequencefor the transcription factor.

Embodiment 79 is the cell of any one of embodiments 75-78, wherein thesecond protein is a protein involved in heme biosynthesis.

Embodiment 80 is the cell of embodiment 79, wherein the protein involvedin heme biosynthesis is selected from the group consisting ofaminolevulinic acid synthase (ALAS), δ-aminolevulinic acid dehydratase(ALAD), porphogilinogen deaminase (PBGD), uroporphyrinogen III synthase(UPG3 S), uroporphyrinogen III decarboxylase (UPG3D),coprotoporphyrinogen oxidase (COPROX), protoporphyrinogen IX oxidase(PROTOX), and ferrochelatase (FC).

Embodiment 81 is a method of producing a product in a cell comprising:

expressing a nucleic acid construct comprising a nucleotide sequenceoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element comprises a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions668-734 relative to SEQ ID NO: 28.

Embodiment 82 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises a mutation at one or more nucleotidepositions corresponding to any of nucleotide positions 673-729 relativeto SEQ ID NO: 28.

Embodiment 83 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises a mutation at one or more nucleotidepositions corresponding to any of nucleotide positions 678-724 relativeto SEQ ID NO: 28.

Embodiment 84 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises a mutation at one or more nucleotidepositions corresponding to any of nucleotide positions 683-719 relativeto SEQ ID NO: 28.

Embodiment 85 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises a mutation at one or more nucleotidepositions corresponding to any of nucleotide positions 688-714 relativeto SEQ ID NO: 28.

Embodiment 86 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises two or more mutations at nucleotidepositions corresponding to any of nucleotide positions 668-734 relativeto SEQ ID NO: 28.

Embodiment 87 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises three or more mutations at nucleotidepositions corresponding to any of nucleotide positions 668-734 relativeto SEQ ID NO: 28.

Embodiment 88 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises four or more mutations at nucleotidepositions corresponding to any of nucleotide positions 668-734 relativeto SEQ ID NO: 28.

Embodiment 89 is the method of embodiment 81, wherein the first alcoholoxidase promoter element comprises five or more mutations at nucleotidepositions corresponding to any of nucleotide positions 668-734 relativeto SEQ ID NO: 28.

Embodiment 90 is the method of any one of embodiments 81-89, wherein atiter of the product produced by expressing a nucleic acid constructcomprising a nucleotide sequence operably linked to a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises a mutation at one or more nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO: 28 is greater than a titer of the product produced expressing anucleic acid construct comprising a nucleotide sequence operably linkedto a first alcohol oxidase promoter element, wherein the first alcoholoxidase promoter element lacks any mutation at nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO: 28.

Embodiment 91 is a method of producing a product in a cell comprising:

expressing a nucleic acid construct comprising a nucleotide sequenceoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element comprises one or more mutationsat a nucleotide position selected from the group consisting ofnucleotide positions corresponding to T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO: 28.

Embodiment 92 is the method of embodiment 91, wherein the first alcoholoxidase promoter element comprises two or more mutations at nucleotidepositions selected from the group consisting of nucleotide positionscorresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596,T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relativeto SEQ ID NO: 28.

Embodiment 93 is the method of embodiment 91, wherein the first alcoholoxidase promoter element includes three or more mutations at nucleotidepositions selected from the group consisting of nucleotide positionscorresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596,T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relativeto SEQ ID NO: 28.

Embodiment 94 is the method of embodiment 91, wherein the first alcoholoxidase promoter element includes four or more mutations at nucleotidepositions selected from the group consisting of nucleotide positionscorresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596,T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relativeto SEQ ID NO: 28.

Embodiment 95 is the method of embodiment 91, wherein the first alcoholoxidase promoter element includes five or more mutations at nucleotidepositions selected from the group consisting of nucleotide positionscorresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596,T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relativeto SEQ ID NO: 28.

Embodiment 96 is the method of any one of embodiments 91-95, wherein thefirst alcohol oxidase promoter element includes one or more mutations ata nucleotide position selected from the group consisting of nucleotidepositions corresponding to of T688, A696, T702, A712, and T714 relativeto SEQ ID NO: 28.

Embodiment 97 is the method of any one of embodiments 91-95, wherein thefirst alcohol oxidase promoter element includes two or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to of T688, A696, T702, A712, and T714 relativeto SEQ ID NO: 28.

Embodiment 98 is the method of any one of embodiments 91-95, wherein thefirst alcohol oxidase promoter element includes three or more mutationsat nucleotide positions selected from the group consisting of nucleotidepositions corresponding to of T688, A696, T702, A712, and T714 relativeto SEQ ID NO: 28.

Embodiment 99 is the method of any one of embodiments 91-95, wherein thefirst alcohol oxidase promoter element includes four or more mutationsat nucleotide positions selected from the group consisting of nucleotidepositions corresponding to of T688, A696, T702, A712, and T714 ascompared to SEQ ID NO: 28.

Embodiment 100 is the method of any one of embodiments 91-95, whereinthe first alcohol oxidase promoter element includes mutations atnucleotide positions corresponding to T688, A696, T702, A712, and T714as compared to SEQ ID NO: 28.

Embodiment 101 is the method of any one of embodiments 91-100, wherein atiter of the product produced by expressing a nucleic acid constructcomprising a nucleotide sequence operably linked to a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises one or more mutations at a nucleotide positionselected from the group consisting of nucleotide positions correspondingto T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688,A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ IDNO: 28 is greater than a titer of the product produced by expressing anucleic acid construct comprising a nucleotide sequence encoding a firstprotein operably linked to a first alcohol oxidase promoter element,wherein the first alcohol oxidase promoter element lacks any mutationsat a nucleotide position selected from the group consisting ofnucleotide positions corresponding to T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO: 28.

Embodiment 102 is a method of producing a product in a cell comprising:

expressing a nucleic acid construct comprising a nucleotide sequenceoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element comprises one or more mutationsat a nucleotide position selected from the group consisting ofnucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530,572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relativeto SEQ ID NO: 28.

Embodiment 103 is the method of embodiment 102, wherein the firstalcohol oxidase promoter element comprises two or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596,617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relative to SEQ IDNO: 28.

Embodiment 104 is the method of embodiment 102, wherein the firstalcohol oxidase promoter element includes three or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596,617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relative to SEQ IDNO: 28.

Embodiment 105 is the method of embodiment 102, wherein the firstalcohol oxidase promoter element includes four or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596,617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relative to SEQ IDNO: 28.

Embodiment 106 is the method of embodiment 102, wherein the firstalcohol oxidase promoter element includes five or more mutations atnucleotide positions selected from the group consisting of nucleotidepositions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596,617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 relative to SEQ IDNO: 28.

Embodiment 107 is the method of any one of embodiments 102-106, whereinthe first alcohol oxidase promoter element includes one or moremutations at a nucleotide position selected from the group consisting ofnucleotide positions corresponding to of 688, 696, 702, 712, and 714relative to SEQ ID NO: 28.

Embodiment 108 is the method of any one of embodiments 102-106, whereinthe first alcohol oxidase promoter element includes two or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to of 688, 696, 702, 712, and 714relative to SEQ ID NO: 28.

Embodiment 109 is the method of any one of embodiments 102-106, whereinthe first alcohol oxidase promoter element includes three or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to of 688, 696, 702, 712, and 714relative to SEQ ID NO: 28.

Embodiment 110 is the method of any one of embodiments 102-106, whereinthe first alcohol oxidase promoter element includes four or moremutations at nucleotide positions selected from the group consisting ofnucleotide positions corresponding to of 688, 696, 702, 712, and 714 ascompared to SEQ ID NO: 28.

Embodiment 111 is the method of any one of embodiments 102-106, whereinthe first alcohol oxidase promoter element includes mutations atnucleotide positions corresponding to 688, 696, 702, 712, and 714 ascompared to SEQ ID NO: 28.

Embodiment 112 is the method of any one of embodiments 102-111, whereina titer of the product produced by expressing a nucleic acid constructcomprising a nucleotide sequence operably linked to a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement comprises one or more mutations at a nucleotide positionselected from the group consisting of nucleotide positions correspondingto 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709,712, 714, 790, 841, and 862 relative to SEQ ID NO: 28 is greater than atiter of the product produced by expressing a nucleic acid constructcomprising a nucleotide sequence encoding a first protein operablylinked to a first alcohol oxidase promoter element, wherein the firstalcohol oxidase promoter element lacks any mutations at a nucleotideposition selected from the group consisting of nucleotide positionscorresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688,696, 702, 709, 712, 714, 790, 841, and 862 relative to SEQ ID NO: 28.

Embodiment 113 is a method of producing a product in a cell comprising:

expressing a nucleic acid construct comprising a nucleotide sequenceoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element includes one or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C,A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative toSEQ ID NO: 28.

Embodiment 114 is the method of embodiment 113, wherein the firstalcohol oxidase promoter element includes two or more mutations selectedfrom the group consisting of mutations corresponding to T146C, C154T,T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T,T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ IDNO: 28.

Embodiment 115 is the method of embodiment 113, wherein the firstalcohol oxidase promoter element includes three or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C,A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative toSEQ ID NO: 28.

Embodiment 116 is the method of embodiment 113, wherein the firstalcohol oxidase promoter element includes four or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C,A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative toSEQ ID NO: 28.

Embodiment 117 is the method of embodiment 113, wherein the firstalcohol oxidase promoter element includes five or more mutationsselected from the group consisting of mutations corresponding to T146C,C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C,A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative toSEQ ID NO: 28.

Embodiment 118 is the method of any one of embodiments 113-117, whereinthe titer of a product produced by expressing a nucleic acid constructcomprising a nucleotide sequence operably linked to a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement includes one or more mutations selected from the groupconsisting of mutations corresponding to T146C, C154T, T303C, T426A,A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G,A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28 isgreater than the titer of a product produced by expressing a nucleicacid construct comprising a nucleotide sequence encoding a first proteinoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element lacks any mutations selected fromthe group consisting of mutations corresponding to T146C, C154T, T303C,T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C,A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28.

Embodiment 119 is the method of any one of embodiments 81-118, whereinthe first alcohol oxidase promoter element comprises two or moremutations selected from the group consisting of T688C, A696T, T702C,A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 120 is the method of any one of embodiments 81-118, whereinthe first alcohol oxidase promoter element comprises three or moremutations selected from the group consisting of T688C, A696T, T702C,A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 121 is the method of any one of embodiments 81-118, whereinthe first alcohol oxidase promoter element comprises four or moremutations selected from the group consisting of T688C, A696T, T702C,A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 122 is the method of any one of embodiments 81-118, whereinthe first alcohol oxidase promoter element comprises the mutationsT688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO: 28.

Embodiment 123 is a method of producing a product in a cell comprising:

expressing a nucleic acid construct comprising a nucleotide sequenceoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element includes one or more mutationsselected from the group consisting of mutations corresponding to 146C,154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C,709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ ID NO: 28.

Embodiment 124 is the method of embodiment 123, wherein the firstalcohol oxidase promoter element includes two or more mutations selectedfrom the group consisting of mutations corresponding to 146C, 154T,303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G,712G, 714G, 790G, 841T, and 862A relative to SEQ ID NO: 28.

Embodiment 125 is the method of embodiment 123, wherein the firstalcohol oxidase promoter element includes three or more mutationsselected from the group consisting of mutations corresponding to 146C,154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C,709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ ID NO: 28.

Embodiment 126 is the method of embodiment 123, wherein the firstalcohol oxidase promoter element includes four or more mutationsselected from the group consisting of mutations corresponding to 146C,154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C,709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ ID NO: 28.

Embodiment 127 is the method of embodiment 123, wherein the firstalcohol oxidase promoter element includes five or more mutationsselected from the group consisting of mutations corresponding to 146C,154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C,709G, 712G, 714G, 790G, 841T, and 862A relative to SEQ ID NO: 28.

Embodiment 128 is the method of any one of embodiments 123-127, whereinthe titer of a product produced by expressing a nucleic acid constructcomprising a nucleotide sequence operably linked to a first alcoholoxidase promoter element, wherein the first alcohol oxidase promoterelement includes one or more mutations selected from the groupconsisting of mutations corresponding to 146C, 154T, 303C, 426A, 433T,435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G,841T, and 862A relative to SEQ ID NO: 28 is greater than the titer of aproduct produced by expressing a nucleic acid construct comprising anucleotide sequence encoding a first protein operably linked to a firstalcohol oxidase promoter element, wherein the first alcohol oxidasepromoter element lacks any mutations selected from the group consistingof mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A,572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and862A relative to SEQ ID NO: 28.

Embodiment 129 is the method of any one of embodiments 81-128, whereinthe first alcohol oxidase promoter element comprises two or moremutations selected from the group consisting of 688C, 696T, 702C, 712G,and 714G relative to SEQ ID NO: 28.

Embodiment 130 is the method of any one of embodiments 81-128, whereinthe first alcohol oxidase promoter element comprises three or moremutations selected from the group consisting of 688C, 696T, 702C, 712G,and 714G relative to SEQ ID NO: 28.

Embodiment 131 is the method of any one of embodiments 81-128, whereinthe first alcohol oxidase promoter element comprises four or moremutations selected from the group consisting of 688C, 696T, 702C, 712G,and 714G relative to SEQ ID NO: 28.

Embodiment 132 is the method of any one of embodiments 81-128, whereinthe first alcohol oxidase promoter element comprises the mutations 688C,696T, 702C, 712G, and 714G relative to SEQ ID NO: 28.

Embodiment 133 is a method of producing a product in a cell comprising:

expressing a nucleic acid construct comprising a nucleotide sequenceoperably linked to a first alcohol oxidase promoter element, wherein thefirst alcohol oxidase promoter element is the nucleic acid construct ofany one of embodiments 1-54.

Embodiment 134 is the method of any one of embodiments 81-133, whereinthe first alcohol oxidase promoter element is an alcohol oxidasepromoter element from a promoter selected from the group consisting ofAOX1, AOX2, AOD1, MOX, MOD1, and MOD2.

Embodiment 135 is the method of any one of embodiments 81-134, whereinthe first alcohol oxidase promoter element is an alcohol oxidase 1(AOX1) promoter element.

Embodiment 136 is the method of any one of embodiments 81-135, whereinthe first alcohol oxidase promoter element has at least 90% sequenceidentity to SEQ ID NO: 28.

Embodiment 137 is the method of any one of embodiments 55-135, whereinthe first alcohol oxidase promoter element has at least 95% sequenceidentity to SEQ ID NO: 28.

Embodiment 138 is the method of any one of embodiments 81-137, whereinthe first alcohol oxidase promoter element has the sequence of SEQ IDNO: 29.

Embodiment 139 is the method of any one of embodiments 81-138, whereinthe cell is a yeast cell.

Embodiment 140 is the method of embodiment 139, wherein the yeast cellis a methylotrophic yeast cell.

Embodiment 141 is the method of any one of embodiments 81-140, whereinthe nucleotide sequence operably linked to the first alcohol oxidasepromoter element encodes a first protein.

Embodiment 142 is the method of embodiment 141, wherein the firstprotein is exogenous to the cell.

Embodiment 143 is the method of any one of embodiments 141-142, whereinthe first protein is heterologous to the cell.

Embodiment 144 is the method of any one of embodiments 141-143, whereinthe first protein is selected from the group consisting of an antibodyor fragment thereof, an enzyme, a regulatory protein, a peptide hormone,a blood clotting protein, a cytokine, and a heme-binding protein.

Embodiment 145 is the method of any one of embodiments 141-144, whereinthe first protein is a heme-binding protein.

Embodiment 146 is the method of embodiment 145, wherein the heme-bindingprotein is selected from the group consisting of a globin, a cytochrome,a cytochrome c oxidase, a ligninase, a catalase, and a peroxidase.

Embodiment 147 is the method of embodiment 145, wherein the heme-bindingprotein is selected from the group consisting of an androglobin, achlorocruorin, a cytoglobin, an erythrocruorin, a flavohemoglobin, aglobin E, a globin X, a globin Y, a hemoglobin, a histoglobin, aleghemoglobin, a myoglobin, a neuroglobin, a non-symbiotic hemoglobin, aprotoglobin, and a truncated hemoglobin.

Embodiment 148 is the method of embodiment 145, wherein the heme-bindingprotein is a non-symbiotic hemoglobin.

Embodiment 149 is the method of embodiment 145, wherein the heme-bindingprotein is a leghemoglobin.

Embodiment 150 is the method of embodiment 145, wherein the heme-bindingprotein comprises an amino acid sequence having at least 90% sequenceidentity to an amino acid sequence in any one of SEQ ID NOs: 1-27.

Embodiment 151 is the method of any one of embodiments 81-150, whereinthe first alcohol oxidase promoter element contains one or morerecognition sequences for a transcription factor.

Embodiment 152 is the method of any one of embodiments 81-151, furthercomprising expressing a second nucleic acid construct comprising anucleotide sequence, wherein the nucleotide sequence of the secondnucleic acid construct is operably linked to the first alcohol oxidasepromoter element or to a second promoter element.

Embodiment 153 is the method of embodiment 152, wherein the nucleotidesequence of the second nucleic acid construct is operably linked to asecond promoter element that has the same sequence as the first alcoholoxidase promoter element.

Embodiment 154 is the method of any one of embodiments 152-153, whereinthe nucleotide sequence of the second nucleic acid construct encodes asecond protein.

Embodiment 155 is the method of embodiment 154, wherein the secondprotein is a transcription factor.

Embodiment 156 is the method of embodiment 155, wherein the nucleotidesequence encoding the second protein is operably linked to a secondpromoter element that comprises a recognition sequence for thetranscription factor.

Embodiment 157 is the method of embodiment 155, wherein the firstalcohol oxidase promoter element comprises a recognition sequence forthe transcription factor. Embodiment 158 is the method of 154, whereinthe second protein is a protein involved in heme biosynthesis.

Embodiment 159 is the method of embodiment 158, wherein the proteininvolved in heme biosynthesis is selected from the group consisting ofALAS, ALAD, PBGD, UPG3S, UPG3D, COPROX, PROTOX, and FC.

Embodiment 160 is the method of any one of embodiments 81-159, whereinthe method is carried out in the absence of added methanol.

The materials and methods of the disclosure will be further described inthe following Examples, which do not limit the scope the claims.

EXAMPLES Example 1 Polymerase Chain Reaction

Genes of interest were amplified from genomic DNA or plasmid DNAtemplates using Phusion Hi-fidelity DNA polymerase (New EnglandBiolabs). Briefly, 0.6 μM each of forward and reverse primers wereincubated with 10-50 ng of template DNA and 400 μM of nucleotide mix inthe presence of 1-2 U of Phusion DNA polymerase. The reaction conditionswere as follows:

1 cycle Initial Denaturation 98° C. 1 min 25 cycles Denaturation 98° C.10 sec Annealing 20 sec Extension 72° C. 30 sec per kb 1 cycle FinalExtension 72° C. 5 min 1 cycle Hold 4° C. Forever

Example 2 Plasmid Construction by Ligation

50-100 ng of restriction enzyme digested plasmid and 3× molar excess ofPCR amplified inserts were incubated in the presence of T4 DNA ligase(New England Biolabs). Ligation was carried out at 16° C. for greaterthan 2 hr. 2 μl of ligation reaction was transformed into DH10Belectrocompetent E. coli cells.

Example 3 Transformation into E. coli ElectroMax DH10B T1Phage-Resistant Competent Cells

1.5-2 μl of ligation mixture (Example 2) was transformed into 20 μl ofElectroMax DH10B T1 Phage-Resistant Competent Cells (Invitrogen, Cat #12033-015) by electroporation using MicroPulser (BioRad) set at 1.7 kVusing a 1 mm gap cuvette (BioRad, Cat # 165-2089); after a pulse, 1 mlSOC (super optimal broth with catabolite repression) was added to cellsand cells were incubated at 37° C. for 1 h with shaking at 200 rpm. 10μl of recovery mixture was plated on LB (lysogeny broth) agar platescontaining ampicillin at a concentration of 100 μg/ml. Plates wereincubated overnight at 37° C. Plasmids were isolated and purified usinga NUCLEOSPIN® plasmid kit from Macherev-Nagel, according to themanufacturer's instructions.

Example 4 Preparation of P. pastoris Transformation-Competent Cells

Selected strains of P. pastoris were grown to mid-exponential growthphase (about 2 OD) in 25 ml YPD (yeast extract-peptone-dextrose) medium.Cells were collected by centrifugation at 930× g for 15 minutes. Thecell pellet was resuspended in 2 ml of a solution of 80% YPD and 200 mMHEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 6.8. 75μl of 1 M DTT (dithiothreitol) was added. The resuspended cell pelletwas mixed at 100 rpm at 30° C. for 25 minutes. A 40 ml volume of icecold, sterile water was added to the suspension, and the cells werecollected by centrifugation at 1125× g for 15 minutes and placed on ice.The cell pellet was resuspended in 40 ml ice cold water and collected asbefore for two additional wash steps. The cell pellet was thenresuspended in 20 ml of ice cold 1 M sorbitol and collected bycentrifugation as before. The final cell pellet was suspended in 0.3 mlice cold, sterile 1M sorbitol, aliquoted, and frozen at −80° C.

Example 5 Transformation into P. pastoris

50-100 ng of plasmid DNA was transformed into 30 μl of electrocompetentP. pastoris cells using a 1 mm gap GenePulser cuvette (BioRad) with aGenePulser (BioRad) set at 1.15 kV. 1 ml of YPD/1M sorbitol was addedand mixed at a 1:1 ratio to the cells. The cells were allowed to recoverfor 3 h at 30° C. with shaking at 100 rpm. 100 μl of the recoverymixture was plated on a YPD plate containing the appropriate antibiotic(primary transformation plate), and the rest of the transformed cellswere plated on a second YPD plate with the appropriate antibiotic.Plates were incubated at 30° C. for 48 hours. Primary transformationplates were streaked onto additional YPD plates with appropriateantibiotic, and plates were incubated for 48 h at 30° C. Individualclones were patched onto YPD plates with antibiotics and the patcheswere used to grow the strains in shake flasks for further analysis.

Example 6 Construction of AOX1 Promoter-Green Fluorescent ProteinReporter Vectors

Vectors to monitor expression from the AOX1 promoter and mutatedvariants were constructed using the Green Fluorescent Protein (GFP) as areporter protein. The GFP open reading frame was inserted into the pGABvector (See, e.g., U.S. Pat. No. 9,938,327, incorporated herein byreference in its entirety) with the translation start immediatelydownstream of the methanol-inducible alcohol oxidase 1 (AOX1) promoterfrom Pichia pastoris and the translation stop signal immediatelyfollowed by the transcription terminator sequence from the P. pastorisFDH1 gene.

The open reading frame encoding the Dasher GFP variant protein wasamplified by PCR from the pJ1214-03c plasmid vector obtained from DNA2.0Inc. (Newark, Calif.). The Dasher GFP open reading frame was amplifiedfrom pJ1214-03c with primers MxO0560(GAGGGTCTCGGATGACAGCTTTAACTGAAGGGGCC; SEQ ID NO: 30) and MxO0561(GAGGGTCTCGATTATTGGTAAGTGTCGAGATCAACTGCC; SEQ ID NO: 31), which appendedflanking Eco31I/BsaI restriction endonuclease recognition sites.Amplification was accomplished using PCR as described in Example 1.

The amplified Dasher GFP PCR product and the pGAB vector were digestedwith 10 units of FastDigest Eco31I restriction endonuclease(ThermoFisher Scientific) for 1 hour at 37° C. in 1× FastDigest Buffer(ThermoFisher Scientific). The Eco31I-digested amplified Dasher GFPfragment and pGAB vector were separated by electrophoresis on a 1%agarose gel in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA(ethylenediaminetetraacetic acid), pH 8.3) and visualized using SYBRSafe DNA gel stain (Life Technologies, Carlsbad, Calif.). The desiredDNA fragments were excised from the agarose gel and the DNA wasrecovered using the ZYI\4OCLEAN™ Gel DNA Recovery Kit (Zymo Research,Irvine, Calif.).

The Eco31I-digested fragment containing the Dasher GFP open readingframe was introduced into pGAB at an Eco31I site immediately downstreamof the AOX1 promoter by ligation. A mixture containing 72 ng ofEco31I-digested DNA encoding the Dasher GFP open reading frame and 35 ngof Eco31I-digested pGAB was incubated with 400 units of T4 DNA ligase(New England Biolabs) in 1× T4 DNA ligase reaction buffer (50 mMTris-HC1, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5 @ 25° C.) at 16° C.,for 2 hours in a 20 μl reaction. Electrocompetent E. coli DH10B cellswere transformed with 2 μl of the ligation reaction and antibioticresistant transformants were selected on LSB (listeria special broth)agar plates supplemented with 100 μg/μl ampicillin. Plates wereincubated overnight at 37° C. Colonies were screened for the presence ofthe insert by PCR using primers MxO0560 and MxO0561. The sequence of thefinal vector was confirmed by DNA sequencing.

The resulting vector, pMx0369, included the P. pastoris AOX1 promoterfollowed consecutively by the Dasher GFP open reading frame and the P.pastoris FDH1 terminator. These elements were amplified from pMx0369 DNAwith primers MxO0513 (GTGCTAGGATCCAACATCCAAAGACG; SEQ ID NO: 32) andMxO0514 (TTTTTCTAGAACCTTATCAAGATAGCTAGAAATAGAAATGGTTGC; SEQ ID NO: 33)using the polymerase chain reaction as described in Example 1. Theprimers introduced BamHI and XbaI restriction sites to the 5′ and 3′ends, respectively, of the amplified AOX1 promoter-Dasher GFP-FDH1terminator DNA fragment. These restriction sites were used to clone theDasher GFP and the sequences required for its expression into the pIL75episomal vector. The pIL75 vector carries a panARS autonomousreplication sequence (Liachko & Dunham, 2014, FEMS Yeast Res.,14:364-7), which allows for maintenance of the plasmid vector withoutintegration into the genome of the transformed cells, and a kanMX markerfor selection of transformants with the antibiotic G418. Both theamplified Dasher GFP expression DNA fragment and the pIL75 vector DNAwere digested with 10 units of BamHI and 10 units of XbaI restrictionendonucleases (New England Biolabs) in 1× CutSmart buffer (New EnglandBiolabs) for 1 hour at 37° C. The BamHI-XbaI-digested DNA fragments wereseparated by electrophoresis on a 1% agarose gel in 1× TBE buffer,visualized using SYBR Safe DNA gel stain and the desired DNA fragmentswere excised from the agarose gel and the DNA was recovered using theZymoclean Gel DNA Recovery Kit.

The DNA fragment containing the P. pastoris AOX1 promoter, the DasherGFP open reading frame, and the P. pastoris FDH1 terminator wasintroduced into the similarly digested pIL75 vector by ligation. Amixture containing 48 ng of the BamHI-XbaI-digested DNA fragmentcontaining sequences for the expression of Dasher GFP and 15 ng of theBamHI-XbaI-digested pIL75 DNA was incubated with 400 units of T4 DNAligase (New England Biolabs) in 1× T4 DNA ligase reaction buffer in a 20μl reaction at 16° C., for 2 hours. Electrocompetent E. coli DH10B cellswere transformed with 2 μl of the ligation reaction and antibioticresistant transformants were selected on LSB agar plates supplementedwith 100 μg/μl ampicillin. Plates were incubated overnight at 37° C.Colonies were screened for the presence of the insert by PCR usingprimers MxO0513 and MxO0514. The sequence of the final vector wasconfirmed by DNA sequencing. The resulting episomal vector, containingsequences encoding the Dasher GFP variant whose expression is under thecontrol of the AOX1 promoter was designated pMx0379.

Example 7 Construction of Strain MxY0270

The pMx0379 vector, carrying the Dasher GFP reporter under the controlof the AOX1 promoter, was introduced into Pichia pastoris host strainMxY0051 by transformation. The MxY0051 strain is a MutS strain, butcontains no other modification. Transformants were selected, and theplasmid was maintained, by growth on medium containing the antibioticG418. Plates were incubated at 30° C. for 48 hours. Individual cloneswere patched onto YPD plates with G418 antibiotic and the patches wereused to inoculate cultures in subsequent experiments.

Example 8 Error-Prone Mutagenesis of the AOX1 Promoter

The pMx0369 vector was used as a template for error-prone PCRamplification of the AOX1 promoter. Error-prone PCR was carried out asdescribed in McCullum, et al. (2010, Methods in Molecular Biology,634:103-9). The pAOX1 promoter was amplified using primers MxO0569(TCCTGCAGCCCGGGGGATCCAACATCCAAAGA; SEQ ID NO: 34) and MxO0570(CTTCAGTTAAAGCTGTCATCGTTTCGAATAATTAGT; SEQ ID NO: 35) in a reactioncontaining 1 μM of each primer, 50 ng of template DNA, 1 mM dCTP anddTTP, 0.2 mM dATP and dGTP, 5.5 mM MgC12, and 0.5 mM MnC12, and 5U TaqDNA polymerase, in 1× reaction buffer (Invitrogen). The reactionconditions for error-prone amplification were as follows:

1 cycle Initial Denaturation 94° C. 2 min 25 cycles Denaturation 94° C.30 sec Annealing 55° C. 30 sec Extension 72° C. 2 min 1 cycle FinalExtension 72° C. 5 min

The pMx0379 vector, excluding the pAOX1 promoter sequence, was amplifiedunder standard PCR amplification conditions, as described in Example 1,using primers MxO0571 (AACAACTAATTATTCGAAACGATGACAGCTTTAACT; SEQ ID NO:36) and MxO0572 (ACCTTTCGTCTTTGGATGTTGGATCCCCCGGG; SEQ ID NO: 37).

The pAOX1 promoter generated by error-prone amplification and theamplified pMx0379 vector DNA were each separated by electrophoresis on a1% agarose gel in 1×TBE buffer and visualized using SYBR Safe DNA gelstain. The desired DNA fragments were excised from the agarose gel andthe DNA was recovered using the ZYMOCLEAN™ Gel DNA Recovery Kit asdescribed herein. The pAOX1 promoter sequences (600 ng) and vector DNA(200 ng) were assembled using Gibson assembly reactions (New EnglandBiolabs). The assembly reactions were used to transform ElectroMax DH10Bcompetent cells as described in Example 3. After overnight growth on LBagar plates with ampicillin, the transformants were pooled in 50 ml LBliquid medium containing ampicillin at a concentration of 100 μg/ml andgrown for 4 hours at 37° C. with shaking at 250 rpm. Followingoutgrowth, plasmid DNA was recovered using a QIAGEN Plasmid Midi kit(Qiagen Inc.). The resulting DNA consisted of pMx0379 vectors containinga variety of mutated pAOX1 promoter sequences.

Example 9 Screening the pAOX1 Mutant Library

The pAOX1 promoter library, consisting of pAOX1 promoters generated byerror-prone PCR driving expression of a GFP reporter, was introducedinto strain MxY0051 by transformation. Transformants were selected andmaintained by growth on YPD plates containing the antibiotic G418.Plates were incubated at 30° C. for 72 hours and colonies were screenedfor fluorescence using a Li-Cor Odyssey Fc imaging system (Li-CorBiosciences, Lincoln, Nebr.). A colony that showed significantfluorescence on the YPD was identified. This colony was subcultured ontoa fresh YPD plate along with strain MxY0270 as a wild type pAOX1reference, and confirmed to show increased GFP expression relative tothe reference. This strain was designated MxY0279.

Example 10 Recovery of the Mutated Plasmid from MxY0279

Plasmid DNA was recovered from transformed P. pastoris cells byresuspending a MxY0279 colony in a 100 μl volume of lysis buffer (200 mMLi acetate, 1% SDS) and heating the suspension to 70° C. for 5 minutes.DNA was precipitated from the lysate by the addition of 300 μl of 100%ethanol, followed by centrifugation at 15,000× g for 3 minutes. Therecovered material was washed with a 1 ml volume of 70% ethanol,followed by centrifugation. The precipitated DNA was dissolved in a 100μl volume of DNA elution buffer (5 mM Tris/HCl, pH 8.5). A 2 μl volumeof the recovered DNA solution was used to transform ElectroMax DH10Bcompetent cells as described in Example 3, and bacterial transformantswere recovered by plating on LB plates containing ampicillin at aconcentration of 100 μg/ml. Plasmid DNA was isolated from the bacterialtransformants using a QlAprep Spin Miniprep Kit, and the sequence of themutated promoter was determined by sequencing the plasmid DNA using theMxO0569 and MxO0570 primers. The recovered plasmid vector containing themutant pAOX1 promoter driving GFP expression was designated pMx0414. Thesequence is set forth in SEQ ID NO:29 as shown in FIG. 2, where 19mutation sites are double underlined.

Example 11 Confirmation that the Improved GFP Expression in MxY0279Results from pMx0414

The recovered pMx0414 plasmid was transformed into the MxY0051 strain ofP. pastoris, as described in Example 3. The transformants and theMxY0270 control strain were streaked onto YPD agar plates and incubatedat 30° C. for 3 days. The fluorescence from these cells was measuredusing a Li-Cor Odyssey Fc imaging system as described in Example 9. Thetransformants showed significant expression from the mutant pAOX1promoter on YPD medium, which lacked the inducer methanol, while theMxY0270 control strain bearing a plasmid with GFP driven by the wildtype pAOX1 promoter showed greatly reduced or no fluorescence (FIG. 3).These results confirm that improved GFP expression observed in theoriginal MxY0279 strain was due to the mutations in the pMx0414 plasmid,and not the genome of the host strain.

Example 12 Shake Flask Cultivation of Transformants and Measurement ofGFP Expression

The MxY0270 and MxY0279 strains carrying the pMx0379 and pMx0414 GFPexpression plasmids, as described herein, were inoculated into growthmedia (1% yeast extract, 2% peptone, supplemented with 1% glycerol)containing the antibiotic G418, to maintain the plasmids, and grownovernight at 30° C. with shaking at 200 rpm. The next day the overnightcultures were diluted to an OD600 of 0.5-0.7 with YP media supplementedwith 1% dextrose, 1% glycerol, 1% methanol, or both 1% methanol and 1%dextrose. All media contained the G418 antibiotic.

GFP fluorescence was measured in cultures expressing the reporterprotein using a SpectraMax M2 microplate reader and SoftMax Pro 6.1software (Molecular Devices, San Jose, Calif.) at an excitationwavelength of 485 nm, and an emission wavelength of 525 nm. Fluorescencein shake flask cultures was measured at 48 hours after dilution into therelevant carbon source. GFP fluorescence, in relative fluorescence units(RFU) was normalize to the OD of the culture. (See FIG. 4).

Example 13 Evaluation of a Set of Mutations

A combinatorial promoter library containing the mutations present in aplasmid with all 19 mutations in pAOX1 (promoter designated MxG0038;see, e.g., SEQ ID NO: 29), where each of the positions mutated inMxG0038 was either the wild type or mutant nucleotide. A group of fivemutations from the MxG0038 mutant that confer the improved expressionphenotype was identified. A mutant AOX1 promoter containing mutationsT688C, A696T, T702C, A712G, and T714G was designated MxG0220. A portionof the sequences of MxG0038 and MxG0020 are compared in FIG. 5.

The relative expression of GFP using wild-type pAOX1 promoter, a pAOX1promoter containing all 19 mutations (promoter designated MxG0038 instrain MxY965), a pAOX1 promoter containing the selected 5 mutations(promoter designated MxG0220) is shown in FIG. 6.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A nucleic acid construct comprising a firstalcohol oxidase promoter element, wherein the first alcohol oxidasepromoter element comprises a mutation at one or more nucleotidepositions corresponding to any of nucleotide positions 668-734 relativeto SEQ ID NO:
 28. 2. The nucleic acid construct of claim 1, wherein thefirst alcohol oxidase promoter element comprises a mutation at one ormore nucleotide positions corresponding to any of nucleotide positions678-724 relative to SEQ ID NO:
 28. 3. The nucleic acid construct ofclaim 1, wherein the first alcohol oxidase promoter element comprises amutation at one or more nucleotide positions corresponding to any ofnucleotide positions 688-714 relative to SEQ ID NO:
 28. 4. The nucleicacid construct of claim 1, wherein the first alcohol oxidase promoterelement comprises two or more mutations at nucleotide positionscorresponding to any of nucleotide positions 668-734 relative to SEQ IDNO:
 28. 5. A nucleic acid construct comprising a first alcohol oxidasepromoter element, wherein the first alcohol oxidase promoter elementcomprises one or more mutations at a nucleotide position selected fromthe group consisting of nucleotide positions corresponding to T146,C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702,A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO:
 28. 6. Thenucleic acid construct of claim 5, wherein the first alcohol oxidasepromoter element comprises one or more mutations at a nucleotideposition selected from the group consisting of nucleotide positionscorresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO:28.
 7. The nucleic acid construct of claim 5, wherein the first alcoholoxidase promoter element comprises mutations at nucleotide positionscorresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO:28.
 8. The nucleic acid construct of claim 5, wherein the first alcoholoxidase promoter element comprises one or more mutations selected fromthe group consisting of mutations corresponding to T146C, C154T, T303C,T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C,A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28.9. The nucleic acid construct of claim 5, wherein the first alcoholoxidase promoter element comprises one or more mutations selected fromthe group consisting of T688C, A696T, T702C, A712G, and T714G relativeto SEQ ID NO:
 28. 10. The nucleic acid construct claim 5, wherein thefirst alcohol oxidase promoter element is an alcohol oxidase 1 promoterelement.
 11. The nucleic acid construct of claim 5, further comprising anucleotide sequence, wherein the nucleotide sequence is operably linkedto the first alcohol oxidase promoter element.
 12. The nucleic acidconstruct of claim 11, wherein the nucleotide sequence encodes a firstprotein.
 13. The nucleic acid construct of claim 12, wherein the firstprotein is selected from the group consisting of an antibody or fragmentthereof, an enzyme, a regulatory protein, a peptide hormone, a bloodclotting protein, a cytokine, a cytokine inhibitor, and a heme-bindingprotein.
 14. The nucleic acid construct of claim 12, wherein the firstprotein is a heme-binding protein.
 15. A cell comprising a first nucleicacid construct, wherein the first nucleic acid construct is the nucleicacid construct of claim
 5. 16. A method of producing a product in a cellcomprising: expressing a nucleic acid construct comprising a nucleotidesequence operably linked to a first alcohol oxidase promoter element,wherein the first alcohol oxidase promoter element comprises one or moremutations at a nucleotide position selected from the group consisting ofnucleotide positions corresponding to T146, C154, T303, T426, A433,A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790,A841, and T862 relative to SEQ ID NO:
 28. 17. The method of claim 16,wherein the first alcohol oxidase promoter element includes one or moremutations at a nucleotide position selected from the group consisting ofnucleotide positions corresponding to of T688, A696, T702, A712, andT714 relative to SEQ ID NO:
 28. 18. The method of claim 16, wherein thefirst alcohol oxidase promoter element is an alcohol oxidase 1 promoterelement.
 19. The method of claim 16, wherein the nucleotide sequenceoperably linked to the first alcohol oxidase promoter element encodes afirst protein.
 20. The method of claim 16, wherein the method is carriedout in the absence of added methanol.